Human mesothelin chimeric antigen receptors and uses thereof

ABSTRACT

Provided are compositions and methods for treating diseases associated with expression of mesothelin. Also provided are a chimeric antigen receptor (CAR) specific to mesothelin, vectors encoding the same, and recombinant T cells comprising the mesothelin CAR. Further provided are methods of administering a genetically modified T cell expressing a CAR that comprises a mesothelin binding domain.

This application is a continuation of U.S. application Ser. No.15/105,082, filed Jun. 16, 2016, which is a U.S. National Phaseapplication and claims the benefit of priority under 35 U.S.C. § 371 ofInternational Application No. PCT/CN2014/094393, filed Dec. 19, 2014,which claims priority to International Application No.PCT/CN2013/089979, filed Dec. 19, 2013, International Application No.PCT/CN2014/082610, filed Jul. 21, 2014 and International Application No.PCT/CN2014/090509, filed Nov. 6, 2014. The entire contents of each ofthese applications are incorporated herein by reference.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support under grant numberCA138907 and CA120409 awarded by the National Institutes of Health. Thegovernment has certain rights in the invention.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Jun. 16, 2016 isnamed N2067-7033WO4_SL.TXT and is 375,836 bytes in size.

FIELD OF THE INVENTION

The present invention relates generally to the use of T cells engineeredto express a Chimeric Antigen Receptor (CAR) to treat a diseaseassociated with expression of mesothelin.

BACKGROUND OF THE INVENTION

Mesothelin was originally identified by Pastan and colleagues as a tumorassociated antigen due to its limited expression by normal tissues andoverexpression on tumors. Chang K, et al., Cancer Res. 1992;52(1):181-186 and Chang K, et al. ProcNatlAcadSciUSA. 1996;93(1):136-140. The mesothelin gene encodes a precursor 71-kDa proteinthat is processed to yield the 40-kDa protein, mesothelin, which isanchored at the cell membrane by a glycosylphosphatidyl inositol (GPI)linkage and an amino-terminal 31-kDa shed fragment, called megkaryocytepotentiating factor (MPF). Both fragments contain N-glycosylation sites.A soluble splice variant of the 40-kDa carboxyl-terminal fragment called“soluble mesothelin/MPF-related” has been found in the sera of patientswith pancreatic ductal adenocarcinoma (PDA). Johnston, F, et al.Clinical Cancer Research. 2009; 15(21):6511. Mesothelin is currentlybeing explored both as a therapeutic target as well as a bio-marker fordisease activity and therapeutic response. Argani P, et al. Clin CancerRes. 2001; 7(12):3862-3868.

Mesothelin is a differentiation antigen that is also present on normaltissues. Using the mouse anti-human mesothelin antibody K1 that wasdeveloped by the Pastan group, strong K1 reactivity has beendemonstrated within mesothelial cells that line the peritoneal, pleural,and pericardial cavities, although at lower levels than usually seen formalignant tissues. Chang K, et al., Cancer Res. 1992; 52(1):181-186.Weak K1 reactivity has been detected within the Fallopian tubeepithelium, tracheal basal epithelium and tonsils epithelium. Mesothelinhas also been found on all layers of the cornea. Jirsova K, et al.Experimental eye research. 2010; 91(5):623-629. However, K1 reactivityhas not been detected in the majority of normal tissues including theliver, kidneys, spleen, bone marrow, lymph nodes, thymus, cardiacmuscle, tongue, skeletal muscle, skin, cerebral cortex, cerebellum,spinal cord, peripheral nerve, pituitary, adrenal, salivary gland,mammary gland, thyroid, parathyroid, testis, prostate, epididymis,cervical epithelium, lung parenchyma, esophagus, small-bowel epithelium,colon epithelium, bladder epithelium, gall-bladder epithelium. Chang K,et al., Cancer Res. 1992; 52(1):181-186.

Mesothelin is overexpressed in the vast majority of primary pancreaticadenocarcinomas with rare and weak expression seen in benign pancreatictissue. Argani P, et al. Clin Cancer Res. 2001; 7(12):3862-3868.Epithelial malignant pleural mesothelioma (MPM) universally expressesmesothelin while sarcomatoid MPM does not express mesothelin. Mostserous epithelial ovarian carcinomas, and the related primary peritonealcarcinomas, express mesothelin.

Mesothelin is a target of a natural immune response in ovarian cancer,and has been proposed to be a target for cancer immunotherapy. Bracci L,et al. Clin Cancer Res. 2007; 13(2 Pt 1): 644-653; Moschella F, et al.Cancer Res. 2011; 71(10):3528-3539; Gross G, et al. FASEB J. 1992;6(15):3370-3378; Sadelain M, et al. NatRevCancer. 2003; 3(1):35-45; MuulL M, et al. Blood. 2003; 101(7):2563-2569; Yee C, et al. Proc Natl AcadSci USA. 2002; 99(25):16168-16173. The presence of mesothelin-specificCTLs in patients with pancreatic cancer correlates with overallsurvival. Thomas A M, et al. J Exp Med. 2004; 200:297-306. In addition,Pastan and coworkers have used soluble antibody fragments of ananti-mesothelin antibody conjugated to immunotoxins to treat cancerpatients with mesothelin-positive tumors. This approach has demonstratedadequate safety and some clinical activity in pancreatic cancer. HassanR, et al. Cancer Immun. 2007; 7:20 and Hassan R, et al. Clin Cancer Res.2007; 13(17):5144-5149. In ovarian cancer, this therapeutic strategyproduced one minor response by RECIST criteria and stable disease in asecond patient who also had complete resolution of their ascites.

SUMMARY OF THE INVENTION

The invention features, e.g., methods of providing an immune response inpatients by administering an immune effector cell that is engineered toexpress a Chimeric Antigen Receptor (CAR) that comprises an antibody(e.g., scFv) that specifically targets mesothelin. In particular, theinvention pertains to the use of an immune effector cell such as, e.g.,a T cell or NK cell, engineered to express a CAR that includes anantibody such as antigen binding fragment thereof to treat a cancerassociated with expression of mesothelin (or MSLN). In particular, theinvention pertains to adoptive cell transfer that may be particularlysuitable for patients with mesothelin-expressing cancers, such as, e.g.,mesothelioma (e.g., malignant pleural mesothelioma, lung cancer (e.g.,non-small cell lung cancer, small cell lung cancer, squamous cell lungcancer, or large cell lung cancer), pancreatic cancer (e.g., pancreaticductal adenocarcinoma, pancreatic metatstatic), ovarian cancer,colorectal cancer and bladder cancer, or any combination thereof.

Accordingly, in one aspect, the invention pertains to an isolatednucleic acid molecule encoding a chimeric antigen receptor (CAR),wherein the CAR comprises an anti-mesothelin binding domain (e.g., ahuman anti-mesothelin binding domain), a transmembrane domain, and anintracellular signaling domain comprising a stimulatory domain. In oneembodiment, the encoded anti-mesothelin binding domain comprises one ormore (e.g., all three) light chain complementary determining region 1(LC CDR1), light chain complementary determining region 2 (LC CDR2), andlight chain complementary determining region 3 (LC CDR3) of a humananti-mesothelin binding domain described herein, and one or more (e.g.,all three) heavy chain complementary determining region 1 (HC CDR1),heavy chain complementary determining region 2 (HC CDR2), and heavychain complementary determining region 3 (HC CDR3) of a humananti-mesothelin binding domain described herein. In one embodiment, theencoded human anti-mesothelin binding domain comprises, or consists of,a light chain variable region described herein (e.g., in Table 2, 4 or5) and/or a heavy chain variable region described herein (e.g., in Table2, 4, or 5). In one embodiment, the encoded anti-mesothelin bindingdomain is a scFv comprising or consisting of a light chain and a heavychain of an amino acid sequence of Table 2. In an embodiment, theanti-mesothelin binding domain (e.g., an scFV) comprises or consists: alight chain variable region comprising an amino acid sequence having atleast one, two or three modifications (e.g., substitutions) but not morethan 30, 20 or 10 modifications (e.g., substitutions) of an amino acidsequence of a light chain variable region provided in Table 2, or asequence with 95-99% identity with an amino acid sequence of Table 2;and/or a heavy chain variable region comprising, or consisting of, anamino acid sequence having at least one, two or three modifications(e.g., substitutions) but not more than 30, 20 or 10 modifications(e.g., substitutions) of an amino acid sequence of a heavy chainvariable region provided in Table 2, or a sequence with 95-99% identityto an amino acid sequence of Table 2. In one embodiment, the humananti-mesothelin binding domain comprises, or consists of, a sequenceselected from the group consisting of SEQ ID NO: 39; SEQ ID NO: 40, SEQID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45,SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO:50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ IDNO: 55, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59, SEQID NO: 60, SEQ ID NO: 61, and SEQ ID NO: 62, or a sequence with 95-99%identity thereof. In one embodiment, the nucleic acid sequence encodingthe human anti-mesothelin binding domain comprises, or consists of, asequence selected from the group consisting of SEQ ID NO: 87; SEQ ID NO:88, SEQ ID NO: 89, SEQ ID NO: 90, SEQ ID NO: 91, SEQ ID NO: 92, SEQ IDNO: 93, SEQ ID NO: 94, SEQ ID NO: 95, SEQ ID NO: 96, SEQ ID NO: 97, SEQID NO: 98, SEQ ID NO: 99, SEQ ID NO: 100, SEQ ID NO: 101, SEQ ID NO:102, SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID NO: 105, SEQ ID NO: 106, SEQID NO: 107, SEQ ID NO: 108, SEQ ID NO: 109, and SEQ ID NO: 110, or asequence with 95-99% identify thereof.

In one embodiment, the isolated nucleic acid further comprises asequence encoding a transmembrane domain, e.g., a transmembrane domaindescribed herein. In one embodiment, the encoded transmembrane domaincomprises, or consists of, a transmembrane domain of a protein selectedfrom the alpha, beta or zeta chain of the T-cell receptor, CD28, CD3epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80,CD86, CD134, CD137 and CD154. In one embodiment, the encodedtransmembrane domain comprises, or consists of, a sequence of SEQ ID NO:12. In one embodiment, the transmembrane domain comprises, or consistsof, an amino acid sequence having at least one, two or threemodifications (e.g., substitutions) but not more than 20, 10 or 5modifications (e.g., substitutions) of an amino acid sequence of SEQ IDNO: 12, or a sequence with 95-99% identity to an amino acid sequence ofSEQ ID NO: 12.

In one embodiment, the encoded CAR includes an anti-mesothelin bindingdomain, e.g., an anti-mesothelin binding domain described herein,connected to the transmembrane domain by a hinge region, e.g., a hingeregion described herein. In one embodiment, the hinge region comprises,or consists of, SEQ ID NO: 6 or SEQ ID NO: 8.

In one embodiment, the isolated nucleic acid molecule further comprisesa sequence encoding a costimulatory domain, e.g., a costimulatory domaindescribed herein. In one embodiment, the costimulatory domain is afunctional signaling domain obtained from a protein selected from thegroup consisting of OX40, CD27, CD28, CD5, ICAM-1, LFA-1 (CD11a/CD18),ICOS (CD278), and 4-1BB (CD137). In one embodiment, the encodedcostimulatory domain comprises, or consists of, a sequence of SEQ IDNO:14. In one embodiment, the costimulatory domain comprises, orconsists of, an amino acid sequence having at least one, two or threemodifications (e.g., substitutions) but not more than 20, 10 or 5modifications (e.g., substitutions) of an amino acid sequence of SEQ IDNO: 14, or a sequence with 95-99% identity to an amino acid sequence ofSEQ ID NO: 14.

In one embodiment, the isolated nucleic acid comprises a sequenceencoding an intracellular signaling domain, e.g., an intracellularsignaling domain described herein. In one embodiment, the isolatednucleic acid encodes a functional signaling domain of 4-1BB and/or afunctional signaling domain of CD3 zeta. In one embodiment, the encodedintracellular signaling domain comprises the sequence of SEQ ID NO: 7and/or the sequence of SEQ ID NO: 9 or SEQ ID NO: 10. In one embodiment,the intracellular signaling domain comprises an amino acid sequencehaving at least one, two or three modifications (e.g., substitutions)but not more than 20, 10 or 5 modifications (e.g., substitutions) of anamino acid sequence of SEQ ID NO:7 and/or an amino acid sequence of SEQID NO:9 or SEQ ID NO: 10, or a sequence with 95-99% identity to an aminoacid sequence of SEQ ID NO:7 and/or an amino acid sequence of SEQ IDNO:9 or SEQ ID NO: 10. In one embodiment, the encoded intracellularsignaling domain comprises, or consists of, the sequence of SEQ ID NO: 7and the sequence of SEQ ID NO: 9 or SEQ ID NO: 10, wherein the sequencescomprising the intracellular signaling domain are expressed in the sameframe and as a single polypeptide chain.

In another aspect, the invention pertains to an isolated nucleic acidmolecule encoding a CAR construct comprising a leader sequence, e.g., ofSEQ ID NO: 1; an anti-mesothelin binding domain described herein, e.g.,having an amino acid sequence of Table 2, or a sequence with 95-99%identify thereof; a hinge region, e.g., of SEQ ID NO: 2; a transmembranedomain, e.g., having a sequence of SEQ ID NO: 6; a costimulatory domain,e.g., a 4-1BB costimulatory domain having a sequence of SEQ ID NO:7; anda primary signaling domain, e.g., CD3 zeta stimulatory domain having asequence of SEQ ID NO:9 or 10. In one embodiment, the isolated nucleicacid molecule comprises (e.g., consists of) a nucleic acid sequenceencoding a polypeptide having an amino acid sequence of Table 2. In oneembodiment, the isolated nucleic acid molecule comprises (consists of) anucleic acid encoding a polypeptide having an amino acid sequence havingat least one, two or three modifications (e.g., substitutions) but notmore than 30, 20 or 10 modifications (e.g., substitutions) of an aminoacid sequence of Table 2, or a sequence with 95-99% identity to an aminoacid sequence of Table 2.

In another aspect, the invention pertains to an isolated polypeptidemolecule encoded by the nucleic acid sequence, e.g., a nucleic aciddescribed herein.

In another aspect, the invention pertains to an isolated polypeptidemolecule comprising, or consisting of, a sequence selected from thegroup consisting of Table 2, an amino acid sequence having at least one,two or three modifications (e.g., substitutions) but not more than 30,20 or 10 modifications (e.g., substitutions) of an amino acid sequenceof a heavy chain variable region provided in Table 2, or a sequence with95-99% identity to an amino acid sequence of Table 2. In one embodiment,the isolated polypeptide comprises one or more (e.g., all three) lightchain complementary determining region 1 (LC CDR1), light chaincomplementary determining region 2 (LC CDR2), and light chaincomplementary determining region 3 (LC CDR3) of a human anti-mesothelinbinding domain described herein, and one or more (e.g., all three) heavychain complementary determining region 1 (HC CDR1), heavy chaincomplementary determining region 2 (HC CDR2), and heavy chaincomplementary determining region 3 (HC CDR3) of a human anti-mesothelinbinding domain described herein.

In another aspect, the invention pertains to an isolated chimericantigen receptor (CAR) molecule comprising an anti-mesothelin bindingdomain described herein, e.g., a human anti-mesothelin binding domaindescribed herein, a transmembrane domain, and an intracellular signalingdomain comprising a stimulatory domain.

In one embodiment, the anti-mesothelin binding domain does not competefor binding to human mesothelin with an antigen binding domaincomprising a sequence comprising SEQ ID NO: 279.

In one embodiment, the anti-mesothelin binding domain competes forbinding to human mesothelin with an antigen binding domain comprising aLC CDR1, LC CDR2 and LC CDR3 of an anti-mesothelin light chain aminoacid sequence selected from SEQ ID NO: 43 or SEQ ID NO: 49 and an HCCDR1, HC CDR2, and HC CDR3 of an anti-mesothelin heavy chain amino acidsequence selected from SEQ ID NO: 43 or SEQ ID NO: 49. In oneembodiment, the anti-mesothelin binding domain competes for binding tohuman mesothelin with an antigen binding domain comprising SEQ ID NO:43or SEQ ID NO:49.

In one embodiment, the anti-mesothelin binding domain binds to adifferent epitope of human mesothelin than the epitope of humanmesothelin targeted by the antigen binding domain comprising a sequencecomprising SEQ ID NO: 279. In an embodiment, the epitope comprises asequence of amino acids selected from amino acids 314-315, 317-318,346-349, and 369-375 of SEQ ID NO: 278, or any combination thereof. Inan embodiment, the epitope comprises one or more amino acids selectedfrom amino acids 314-315, 317-318, 346-349, and 369-375 of SEQ ID NO:278, or any combination thereof.

In an embodiment, the anti-mesothelin binding domain described hereindoes not bind to the N-terminus of mesothelin as shown in SEQ ID NO:278. In one embodiment, the anti-mesothelin binding domain binds to theC-terminus of human mesothelin. In one embodiment, the anti-mesothelinbinding domain binds an epitope within amino acids 450-588 of SEQ ID NO:278. In one embodiment, the epitope bound by the anti-mesothelin bindingdomain comprises a sequence selected from amino acids 485-490, 498-507,532-537, and 545-572 of SEQ ID NO: 278, or a combination thereof. In oneembodiment, the epitope bound by the anti-mesothelin binding domaincomprises one or more amino acids selected from amino acids 485-490,498-507, 532-537, and 545-572 of SEQ ID NO: 278, or any combinationthereof. In these embodiments, SEQ ID NO: 278 represents amino acids296-588 of human mesothelin, e.g., the first amino acid of SEQ ID NO:278 is amino acid 296 and the last amino acid of SEQ ID NO: 278 is aminoacid 588.

In one embodiment, the anti-mesothelin binding domain comprises one ormore (e.g., all three) light chain complementary determining region 1(LC CDR1), light chain complementary determining region 2 (LC CDR2), andlight chain complementary determining region 3 (LC CDR3) of a humananti-mesothelin binding domain described herein, and one or more (e.g.,all three) heavy chain complementary determining region 1 (HC CDR1),heavy chain complementary determining region 2 (HC CDR2), and heavychain complementary determining region 3 (HC CDR3) of a humananti-mesothelin binding domain described herein. In one embodiment, thehuman anti-mesothelin binding domain comprises, or consists of, a lightchain variable region described herein (e.g., in Table 2) and/or a heavychain variable region described herein (e.g., in Table 2). In oneembodiment, the anti-mesothelin binding domain is a scFv comprising, orconsisting of, a light chain variable region and a heavy chain variableregion of an amino acid sequence of Table 2. In an embodiment, theanti-mesothelin binding domain (e.g., an scFV) comprises, or consistsof: a light chain variable region comprising an amino acid sequencehaving at least one, two or three modifications (e.g., substitutions)but not more than 30, 20 or 10 modifications (e.g., substitutions) of anamino acid sequence of a light chain variable region provided in Table2, or a sequence with 95-99% identity to an amino acid sequence of Table2; and/or a heavy chain variable region comprising an amino acidsequence having at least one, two or three modifications (e.g.,substitutions) but not more than 30, 20 or 10 modifications (e.g.,substitutions) of an amino acid sequence of a heavy chain variableregion provided in Table 2, or a sequence with 95-99% identity to anamino acid sequence of Table 2. In one embodiment, the humananti-mesothelin binding domain comprises, or consists of, a sequenceselected from the group consisting of SEQ ID NO: 39; SEQ ID NO: 40, SEQID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45,SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO:50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ IDNO: 55, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59, SEQID NO: 60, SEQ ID NO: 61, and SEQ ID NO: 62, or a sequence with 95-99%identity thereof.

In one embodiment, the transmembrane domain is a transmembrane domain ofa protein selected from the group consisting of the alpha, beta or zetachain of the T-cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8,CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137 and CD154.In one embodiment, the transmembrane domain comprises a transmembranedomain described herein, e.g., having a sequence of SEQ ID NO:6, anamino acid sequence having at least one, two or three modifications(e.g., substitutions) but not more than 20, 10 or 5 modifications (e.g.,substitutions) of an amino acid sequence of SEQ ID NO:6, or a sequencewith 95-99% identity to an amino acid sequence of SEQ ID NO:6.

In one embodiment, the anti-mesothelin binding domain is connected tothe transmembrane domain by a hinge region. In one embodiment, the hingeregion comprises a hinge region described herein, e.g., a hinge regionof SEQ ID NO:2.

In one embodiment, the isolated CAR molecule further comprises acostimulatory domain, e.g., a costimulatory domain described herein. Inone embodiment, the costimulatory domain is a functional signalingdomain obtained from a protein selected from the group consisting ofOX40, CD27, CD28, CD5, ICAM-1, LFA-1 (CD11a/CD18), ICOS (CD278), and4-1BB (CD137) or functional variant thereof. In one embodiment, thecostimulatory domain comprises, or consists of, a sequence of SEQ IDNO:7. In one embodiment, the costimulatory domain comprises, or consistsof, an amino acid sequence having at least one, two or threemodifications (e.g., substitutions) but not more than 20, 10 or 5modifications (e.g., substitutions) of an amino acid sequence of SEQ IDNO:7, or a sequence with 95-99% identity to an amino acid sequence ofSEQ ID NO:7.

In one embodiment, the isolated CAR molecule comprises an intracellularsignaling domain, e.g., an intracellular signaling domain describedherein. In one embodiment, the intracellular signaling domain comprisesa functional signaling domain of 4-1BB and/or a functional signalingdomain of CD3 zeta. In one embodiment, the intracellular signalingdomain comprises, or consists of, the sequence of SEQ ID NO: 7 and/orthe sequence of SEQ ID NO: 9 or SEQ ID NO: 10. In one embodiment, theintracellular signaling domain comprises, or consists of, an amino acidsequence having at least one, two or three modifications (e.g.,substitutions) but not more than 20, 10 or 5 modifications (e.g.,substitutions) of an amino acid sequence of SEQ ID NO:7 and/or an aminoacid sequence of SEQ ID NO:9 or SEQ ID NO: 10, or a sequence with 95-99%identity to an amino acid sequence of SEQ ID NO:7 and/or an amino acidsequence of SEQ ID NO:9 or SEQ ID NO: 10. In one embodiment, theintracellular signaling domain comprises, or consists of, the sequenceof SEQ ID NO: 9 and the sequence of SEQ ID NO: 9 or SEQ ID NO: 10,wherein the sequences comprising the intracellular signaling domain areexpressed in the same frame and as a single polypeptide chain.

In another aspect, the invention pertains to an isolated CAR moleculecomprising a leader sequence, e.g., of SEQ ID NO: 1; an anti-mesothelinbinding domain described herein, e.g., having an amino acid sequence ofTable 2, or a sequence with 95-99% identify thereof; a hinge region,e.g., of SEQ ID NO:2; a transmembrane domain, e.g., having a sequence ofSEQ ID NO: 6; a costimulatory domain, e.g., a 4-1BB costimulatory domainhaving a sequence of SEQ ID NO:7; and a primary signaling domain, e.g.,CD3 zeta stimulatory domain having a sequence of SEQ ID NO:9 or SEQ IDNO: 10. In one embodiment, the isolated CAR molecule comprises (e.g.,consists of) a polypeptide having an amino acid sequence of Table 2. Inone embodiment, the isolated CAR molecule comprises (consists of) apolypeptide having an amino acid sequence having at least one, two orthree modifications (e.g., substitutions) but not more than 30, 20 or 10modifications (e.g., substitutions) of an amino acid sequence of Table2, or a sequence with 95-99% identity to an amino acid sequence of Table2. In one embodiment, the isolated CAR molecule comprises, or consistsof, an amino acid sequence selected from the group consisting of SEQ IDNO: 63; SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQID NO: 68, SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO: 71, SEQ ID NO: 72,SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 75, SEQ ID NO: 76, SEQ ID NO:77, SEQ ID NO: 78, SEQ ID NO: 79, SEQ ID NO: 80, SEQ ID NO: 81, SEQ IDNO: 82, SEQ ID NO: 83, SEQ ID NO: 84, SEQ ID NO: 85, and SEQ ID NO: 86.

In another aspect, the invention pertains to a vector comprising anucleic acid sequence described herein. In one embodiment, the nucleicacid sequence encodes a CAR molecule, e.g., a CAR molecule describedherein. In one embodiment, the vector is selected from the groupconsisting of a DNA, a RNA, a plasmid, a lentivirus vector, adenoviralvector, or a retrovirus vector.

In one embodiment, the vector is a lentivirus vector, e.g., a lentivirusvector described herein. In one embodiment, the vector further comprisesa promoter. In one embodiment, the promoter is an EF-1α promoter. In oneembodiment, the EF-1α promoter comprises a sequence of SEQ ID NO: 11.

In one embodiment, the vector is an in vitro transcribed vector, e.g., avector that transcribes RNA of a nucleic acid molecule described herein.In one embodiment, the RNA is transcribed from an in vitro transcriptionvector, wherein the vector is pD-A.anti-meso BD OF.2bg.150A, wherein theanti-meso BD is an anti-mesothelin binding domain described herein. Inone embodiment, the nucleic acid sequence in the vector furthercomprises a poly(A) tail, e.g., a poly A tail described herein, e.g.,comprising about 150 adenosine bases (SEQ ID NO: 271). In oneembodiment, the nucleic acid sequence in the vector further comprises a3′UTR, e.g., a 3′UTR described herein, e.g., comprising at least onerepeat of a 3′UTR derived from human beta-globulin.

In another aspect, the invention pertains to a cell comprising thevector. The cell can be, e.g., a cell described herein. In oneembodiment, the cell is a human T cell, e.g., a T cell described herein,or a human NK cell, e.g., a human NK cell described herein. In oneembodiment, the human T cell is a CD8+ T cell. In one embodiment, thecell is an autologous T cell. In one embodiment, the cell is anallogeneic T cell. In one embodiment, the cell is a T cell and the Tcell is diaglycerol kinase (DGK) deficient. In one embodiment, the cellis a T cell and the T cell is Ikaros deficient. In one embodiment, thecell is a T cell and the T cell is both DGK and Ikaros deficient.

In one aspect, the CAR-expressing cell described herein can furthercomprise a second CAR, e.g., a second CAR that includes a differentantigen binding domain, e.g., to the same target (mesothelin) or adifferent target (e.g., a target other than mesothelin on stroma cells,e.g., FAP; a target other than mesothelin on prostate cancer cells,e.g., androgen receptor, OR51E2, PSMA, PSCA, PDGRF-β, TARP, GloboH,MAD-CT-1, or MAD-CT-2; a target other than mesothelin on ovarariancancer cells, e.g., Tn, PRSS21, CD171, Lewis Y, folate receptor α,claudin6, GloboH, or sperm protein 17; e.g., a target other thanmesothelin on lung cancer cells, e.g., VEGF, HER3, IGF-1R, EGFR, DLL4,or Trop-2). In one embodiment, the CAR-expressing cell comprises a firstCAR that targets a first antigen and includes an intracellular signalingdomain having a costimulatory signaling domain but not a primarysignaling domain, and a second CAR that targets a second, different,antigen and includes an intracellular signaling domain having a primarysignaling domain but not a costimulatory signaling domain. In oneembodiment, the CAR expressing cell comprises a first mesothelin CARthat includes a mesothelin binding domain, a transmembrane domain and acostimulatory domain and a second CAR that targets an antigen other thanmesothelin (e.g., an antigen expressed on stroma cells, lung cancercells, prostate cancer cells or ovarian cancer cells) and includes anantigen binding domain, a transmembrane domain and a primary signalingdomain. In another embodiment, the CAR expressing cell comprises a firstmesothelin CAR that includes a mesothelin binding domain, atransmembrane domain and a primary signaling domain and a second CARthat targets an antigen other than mesothelin (e.g., an antigenexpressed on stroma cells, lung cancer cells, prostate cancer cells orovarian cancer cells) and includes an antigen binding domain to theantigen, a transmembrane domain and a costimulatory signaling domain.

In one embodiment, the CAR-expressing cell comprises a mesothelin CARdescribed herein and an inhibitory CAR. In one embodiment, theinhibitory CAR comprises an antigen binding domain that binds an antigenfound on normal cells but not cancer cells, e.g., normal cells that alsoexpress mesothelin. In one embodiment, the inhibitory CAR comprises theantigen binding domain, a transmembrane domain and an intracellulardomain of an inhibitory molecule. For example, the intracellular domainof the inhibitory CAR can be an intracellular domain of PD1, PD-L1,CTLA4, TIM3, CEACAM (e.g., CEACAM-1, CEACAM-3 and/or CEACAM-5), LAG3,VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4 and TGFR beta.

In another embodiment, the CAR-expressing cell described herein canfurther express another agent, e.g., an agent which enhances theactivity or fitness of a CAR-expressing cell, e.g., an agent describedherein. For example, in one embodiment, the agent can be an agent whichinhibits a molecule that modulates or regulates, e.g., inhibits, T cellfunction. In some embodimemts, the molecule that modulates or regulatesT cell function is an inhibitory molecule. Examples of inhibitorymolecules include PD1, PD-L1, CTLA4, TIM3, CEACAM (e.g., CEACAM-1,CEACAM-3 and/or CEACAM-5), LAG3, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4and TGFR beta. In embodiments, an agent, e.g., an inhibitory nucleicacid, e.g., a dsRNA, e.g., an siRNA or shRNA; or e.g., an inhibitoryprotein or system, e.g., a clustered regularly interspaced shortpalindromic repeats (CRISPR), a transcription-activator like effectornuclease (TALEN), or a zinc finger endonuclease (ZFN), e.g., asdescribed herein, can be used to inhibit expression of a molecule thatmodulates or regulates, e.g., inhibits, T-cell function in theCAR-expressing cell. In an embodiment the agent is an shRNA, e.g., anshRNA described herein. In an embodiment, the agent that modulates orregulates, e.g., inhibits, T-cell function is inhibited within aCAR-expressing cell. For example, a dsRNA molecule that inhibitsexpression of a molecule that modulates or regulates, e.g., inhibits,T-cell function is linked to the nucleic acid that encodes a component,e.g., all of the components, of the CAR.

In one embodiment, the agent which inhibits an inhibitory moleculecomprises a first polypeptide, e.g., an inhibitory molecule, associatedwith a second polypeptide that provides a positive signal to the cell,e.g., an intracellular signaling domain described herein. In oneembodiment, the agent comprises a first polypeptide, e.g., of aninhibitory molecule such as PD1, PD-L1, CEACAM (e.g., CEACAM-1, CEACAM-3and/or CEACAM-5), LAG3, CTLA4, VISTA, CD160, BTLA, LAIR1, TIM3, 2B4,TGFR beta and TIGIT, or a fragment of any of these (e.g., at least aportion of the extracellular domain of any of these), and a secondpolypeptide which is an intracellular signaling domain described herein(e.g., comprising a costimulatory domain (e.g., 41BB, CD27 or CD28,e.g., as described herein) and/or a primary signaling domain (e.g., aCD3 zeta signaling domain described herein)). In one embodiment, theagent comprises a first polypeptide of PD1 or a fragment thereof (e.g.,at least a portion of the extracellular domain of PD1), and a secondpolypeptide of an intracellular signaling domain described herein (e.g.,a CD28 signaling domain described herein and/or a CD3 zeta signalingdomain described herein).

In another aspect, the invention pertains to a method of making a cellcomprising transducing a cell described herein, e.g., a T cell or a NKcell, with a vector of comprising a nucleic acid encoding a CARmolecule, e.g., a CAR molecule described herein. In one embodiment, thevector is a lentiviral vector described herein.

The present invention also provides a method of generating a populationof RNA-engineered cells, e.g., cells described herein, e.g., T cells orNK cells, transiently expressing exogenous RNA. The method comprisesintroducing an in vitro transcribed RNA or synthetic RNA into a cell,where the RNA comprises a nucleic acid encoding a CAR molecule describedherein.

In another aspect, the invention pertains to a method of providinganti-tumor immunity in a subject comprising administering to the subjectan effective amount of a cell comprising a CAR molecule, e.g., a cellexpressing a CAR molecule described herein, a cell described herein. Inone embodiment, the cell is an autologous T cell or NK cell. In oneembodiment, the cell is an allogeneic T cell or NK cell. In oneembodiment, the subject is a human.

In another aspect, the invention pertains to a method of treating asubject having a disease associated with expression of mesothelin (e.g.,a proliferative disease, a precancerous condition, and a noncancerrelated indication associated with the expression of mesothelin)comprising administering to the subject an effective amount of a cellcomprising a CAR molecule, e.g., as described herein.

In one embodiment, the disease associated with mesothelin is cancer,e.g., a cancer described herein. In one embodiment, the diseaseassociated with mesothelin is selected from the group consisting of:mesothelioma (e.g., malignant pleural mesothelioma), lung cancer (e.g.,non-small cell lung cancer, small cell lung cancer, squamous cell lungcancer, or large cell lung cancer), pancreatic cancer (e.g., pancreaticductal adenocarcinoma), ovarian cancer, colorectal cancer and bladdercancer or any combination thereof. In one embodiment, the disease ispancreatic cancer, e.g., metastatic pancreatic ductal adenocarcinoma(PDA), e.g., in a subject who has progressed on at least one priorstandard therapy. In one embodiment, the disease is mesothelioma (e.g.,malignant pleural mesothelioma), e.g., in a subject who has progressedon at least one prior standard therapy. In one embodiment, the diseaseis ovarian cancer, e.g., serous epithelial ovarian cancer, e.g., in asubject who has progressed after at least one prior regimen of standardtherapy.

In one embodiment, the mesothelin CAR expressing cell, e.g., T cell orNK cell, is administered to a subject that has received a previous doseof melphalan.

In one embodiment, the cells expressing a CAR molecule, e.g., a CARmolecule described herein, are administered in combination with an agentthat enhances the activity or fitness of a cell expressing a CARmolecule, e.g., an agent described herein.

In one embodiment, the cells expressing a CAR molecule, e.g., a CARmolecule described herein, are administered in combination with a low,immune enhancing dose of an mTOR inhibitor. While not wishing to bebound by theory, it is believed that treatment with a low, immuneenhancing, dose (e.g., a dose that is insufficient to completelysuppress the immune system but sufficient to improve immune function) isaccompanied by a decrease in PD-1 positive T cells or an increase inPD-1 negative cells. PD-1 positive T cells, but not PD-1 negative Tcells, can be exhausted by engagement with cells which express a PD-1ligand, e.g., PD-L1 or PD-L2.

In an embodiment this approach can be used to optimize the performanceof CAR cells described herein in the subject. While not wishing to bebound by theory, it is believed that, in an embodiment, the performanceof endogenous, non-modified immune effector cells, e.g., T cells, isimproved. While not wishing to be bound by theory, it is believed that,in an embodiment, the performance of a mesothelin CAR expressing cell isimproved. In other embodiments, cells, e.g., T cells or NK cells, whichhave, or will be engineered to express a CAR, can be treated ex vivo bycontact with an amount of an mTOR inhibitor that increases the number ofPD1 negative immune effector cells, e.g., T cells or increases the ratioof PD1 negative immune effector cells, e.g., T cells/PD1 positive immuneeffector cells, e.g., T cells.

In an embodiment, administration of a low, immune enhancing, dose of anmTOR inhibitor, e.g., an allosteric inhibitor, e.g., RAD001, or acatalytic inhibitor, is initiated prior to administration of an CARexpressing cell described herein, e.g., T cells or NK cells. In anembodiment, the CAR cells are administered after a sufficient time, orsufficient dosing, of an mTOR inhibitor, such that the level of PD1negative immune effector cells, e.g., T cells, or the ratio of PD1negative immune effector cells, e.g., T cells/PD1 positive immuneeffector cells, e.g., T cells, has been, at least transiently,increased.

In an embodiment, the cell, e.g., T cell or NK cell, to be engineered toexpress a CAR, is harvested after a sufficient time, or after sufficientdosing of the low, immune enhancing, dose of an mTOR inhibitor, suchthat the level of PD1 negative immune effector cells, e.g., T cells, orthe ratio of PD1 negative immune effector cells, e.g., T cells/PD1positive immune effector cells, e.g., T cells, in the subject orharvested from the subject has been, at least transiently, increased.

In one embodiment, the cells expressing a CAR molecule, e.g., a CARmolecule described herein, are administered in combination with an agentthat ameliorates one or more side effect associated with administrationof a cell expressing a CAR molecule, e.g., an agent described herein.

In one embodiment, the cells expressing a CAR molecule, e.g., a CARmolecule described herein, are administered in combination with an agentthat treats the disease associated with mesothelin expression, e.g., anagent described herein.

In one embodiment, the cells expressing a CAR molecule, e.g., a CARmolecule described herein, are administered at a dose and/or dosingschedule described herein.

In one embodiment, the cells expressing a CAR molecule, e.g., a CARmolecule described herein, are administered as a first line treatmentfor the disease, e.g., the cancer, e.g., the cancer described herein. Inanother embodiment, the cells expressing a CAR molecule, e.g., a CARmolecule described herein, are administered as a second, third, fourthline treatment for the disease, e.g., the cancer, e.g., the cancerdescribed herein.

In one embodiment, a population of cells described herein isadministered.

In one embodiment, the CAR molecule is introduced into T cells or NKcells, e.g., using in vitro transcription, and the subject (e.g., human)receives an initial administration of cells comprising a CAR molecule,and one or more subsequent administrations of cells comprising a CARmolecule, wherein the one or more subsequent administrations areadministered less than 15 days, e.g., 14, 13, 12, 11, 10, 9, 8, 7, 6, 5,4, 3, or 2 days after the previous administration. In one embodiment,more than one administration of cells comprising a CAR molecule areadministered to the subject (e.g., human) per week, e.g., 2, 3, or 4administrations of cells comprising a CAR molecule are administered perweek. In one embodiment, the subject (e.g., human subject) receives morethan one administration of cells comprising a CAR molecule per week(e.g., 2, 3 or 4 administrations per week) (also referred to herein as acycle), followed by a week of no administration of cells comprising aCAR molecule, and then one or more additional administration of cellscomprising a CAR molecule (e.g., more than one administration of thecells comprising a CAR molecule per week) is administered to thesubject. In another embodiment, the subject (e.g., human subject)receives more than one cycle of cells comprising a CAR molecule, and thetime between each cycle is less than 10, 9, 8, 7, 6, 5, 4, or 3 days. Inone embodiment, the cells comprising a CAR molecule are administeredevery other day for 3 administrations per week. In one embodiment, thecells comprising a CAR molecule are administered for at least two,three, four, five, six, seven, eight or more weeks.

In one aspect, the invention includes a population of autologous orallogenic cells that are transfected or transduced with a vectorcomprising a nucleic acid molecule encoding a mesothelin-CAR molecule,e.g., as described herein. In one embodiment, the vector is a retroviralvector. In one embodiment, the vector is a self-inactivating lentiviralvector as described elsewhere herein. In one embodiment, the vector isdelivered (e.g., by transfecting or electroporating) to a cell, e.g., aT cell or a NK cell, wherein the vector comprises a nucleic acidmolecule encoding a mesothelin CAR molecule as described herein, whichis transcribed as an mRNA molecule, and the mesothelin CAR molecule istranslated from the RNA molecule and expressed on the surface of thecell.

In another aspect, the present invention provides a population ofCAR-expressing cells, e.g., CART cells. In some embodiments, thepopulation of CAR-expressing cells comprises a mixture of cellsexpressing different CARs. For example, in one embodiment, thepopulation of CART cells can include a first cell expressing a CARhaving an anti-mesothelin binding domain described herein, and a secondcell expressing a CAR having a different anti-mesothelin binding domain,e.g., an anti-mesothelin binding domain described herein that differsfrom the anti-mesothelin binding domain in the CAR expressed by thefirst cell. As another example, the population of CAR-expressing cellscan include a first cell expressing a CAR that includes ananti-mesothelin binding domain, e.g., as described herein, and a secondcell expressing a CAR that includes an antigen binding domain to atarget other than mesothelin (e.g., a target other than mesothelin onstroma cells, e.g., FAP; a target other than mesothelin on prostatecancer cells, e.g., androgen receptor, OR51E2, PSMA, PSCA, PDGRF-β,TARP, GloboH, MAD-CT-1, or MAD-CT-2; a target other than mesothelin onovararian cancer cells, e.g., Tn, PRSS21, CD171, Lewis Y, folatereceptor α, claudin6, GloboH, or sperm protein 17; e.g., a target otherthan mesothelin on lung cancer cells, e.g., VEGF, HER3, IGF-1R, EGFR,DLL4, or Trop-2). In one embodiment, the population of CAR-expressingcells includes, e.g., a first cell expressing a CAR that includes aprimary intracellular signaling domain, and a second cell expressing aCAR that includes a secondary signaling domain.

In another aspect, the present invention provides a population of cellswherein at least one cell in the population expresses a CAR having ananti-mesothelin binding domain described herein, and a second cellexpressing another agent, e.g., an agent which enhances the activity orfunction of a CAR-expressing cell. For example, in one embodiment, theagent can be an agent which inhibits a molecule that modulates orregulates, e.g., inhibits, T cell function. In some embodiments, themolecule that modulates or regulates T cell function is an inhibitorymolecule, e.g., an agent described herein. Examples of inhibitorymolecules include PD1, PD-L1, CTLA4, TIM3, CEACAM (e.g., CEACAM-1,CEACAM-3 and/or CEACAM-5), LAG3, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4and TGFR beta. In embodiments, an agent, e.g., an inhibitory nucleicacid, e.g., a dsRNA, e.g., an siRNA or shRNA; or e.g., an inhibitoryprotein or system, e.g., a clustered regularly interspaced shortpalindromic repeats (CRISPR), a transcription-activator like effectornuclease (TALEN), or a zinc finger endonuclease (ZFN), e.g., asdescribed herein, can be used to inhibit expression of a molecule thatmodulates or regulates, e.g., inhibits, T-cell function in theCAR-expressing cell. In an embodiment the agent is an shRNA, e.g., anshRNA described herein. In an embodiment, the agent that modulates orregulates, e.g., inhibits, T-cell function is inhibited within aCAR-expressing cell. For example, a dsRNA molecule that inhibitsexpression of a molecule that modulates or regulates, e.g., inhibits,T-cell function is linked to the nucleic acid that encodes a component,e.g., all of the components, of the CAR.

In one embodiment, the agent which inhibits an inhibitory moleculecomprises a first polypeptide, e.g., an inhibitory molecule, associatedwith a second polypeptide that provides a positive signal to the cell,e.g., an intracellular signaling domain described herein. In oneembodiment, the agent comprises a first polypeptide, e.g., of aninhibitory molecule such as PD1, PD-L1, CEACAM (e.g., CEACAM-1, CEACAM-3and/or CEACAM-5), LAG3, CTLA4, VISTA, CD160, BTLA, LAIR1, TIM3, 2B4,TGFR beta and TIGIT, or a fragment of any of these (e.g., at least aportion of an extracellular domain of any of these), and a secondpolypeptide which is an intracellular signaling domain described herein(e.g., comprising a costimulatory domain (e.g., 41BB, CD27 or CD28,e.g., as described herein) and/or a primary signaling domain (e.g., aCD3 zeta signaling domain described herein). In one embodiment, theagent comprises a first polypeptide of PD1 or a fragment thereof (e.g.,at least a portion of the extracellular domain of PD1), and a secondpolypeptide of an intracellular signaling domain described herein (e.g.,a CD28 signaling domain described herein and/or a CD3 zeta signalingdomain described herein).

In one embodiment, the nucleic acid molecule encoding a mesothelin CARmolecule, e.g., as described herein, is expressed as an mRNA molecule.In one embodiment, the genetically modified mesothelin CAR-expressingcells, e.g., T cells or NK cells, can be generated by transfecting orelectroporating an RNA molecule encoding the desired CARs (e.g., withouta vector sequence) into the cell. In one embodiment, a mesothelin CARmolecule is translated from the RNA molecule once it is incorporated andexpressed on the surface of the recombinant cell.

In another aspect, the invention pertains to the isolated nucleic acidmolecule encoding a CAR molecule, e.g., a CAR molecule described herein,a CAR molecule described herein, a vector comprising a CAR moleculedescribed herein, and/or a cell comprising a CAR molecule describedherein for use as a medicament.

In another aspect, the invention pertains to a the isolated nucleic acidmolecule encoding a CAR molecule described herein, a CAR moleculedescribed herein, a vector comprising a CAR molecule described herein,and/or a cell comprising a CAR molecule described herein for use in thetreatment of a disease expressing mesothelin, e.g., as described herein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic of the pD-A.anti-meso BD.OF.BBZ.2bg.150A plasmid.Figure discloses “150A” as SEQ ID NO: 271.

FIG. 2A and FIG. 2B depict cell manufacturing and treatment schedulesthat can be used. FIG. 2A: Autologous cells are obtained by leukocyteapheresis and T cells are enriched by expansion with anti-CD3/CD28 mAbcoated magnetic beads. Cells are expanded for 8 to 12 days. On the lastday of culture, the beads are removed using a magnetic field and thecells are washed, electroporated with human meso CAR mRNA construct, andcryopreserved in infusible medium. FIG. 2B: Three treatment infusionschedules are depicted. On Schedule 1, patients receive 1×10⁸ human mesobearing CART cells by intravenous (i.v.) infusion on day 0 followed by1×10⁹ human meso bearing CART cells one week later. Safety can bemonitored for a minimum of one month before patients are eligible forSchedule 2. On Schedule 2, patients receive 1×10⁸ human meso bearingCART cells by i.v. infusion three times per week for one week followedby one week of rest and then 1×10⁹ human meso bearing CART cellsadministered three times per week for one week. On Schedule 3, patientsreceive 3×10⁸/m² human meso bearing CART cells by i.v. infusion threetimes per week for three weeks followed by intra-tumoral injection intoa primary lesion of 2×10⁸ human meso bearing CART cells on days +35 and+57.

FIG. 3A and FIG. 3B are graphic representations of cytotoxicity asassayed donor 2 (healthy donor) T cell transduced with mouse SS1 CAR orthe anti-MSLN CARs M1 to M12 of the invention and cultured with eithercontrol K562 cells that do not express MSLN as shown in FIG. 3A, or K562cells transduced to express MSLN (K562-Meso) as shown in FIG. 3B.

FIG. 4A and FIG. 4B are graphs showing the IFNγ secretion of the mouseSS land CD19 CART and the anti-MSLN CARTs upon stimulation by MSLN+cells. FIG. 4A shows reactivity to the transduced cell line K562-Mesoand its MSLN-negative parental line K562. FIG. 4B shows reactivitytoward cancer cells naturally expressing MSLN; the ovarian cancer lineOvcar8 and the pancreatic cancer lines SW1990 and Panc0203.

FIG. 5 shows a clinical trial design for mesothelin CARTs made bytransducing a CAR construct with a lentiviral vector.

FIG. 6A, FIG. 6B, FIG. 6C, and FIG. 6D show anti-tumor activity ofCART-meso cells. FIG. 6A shows the partial response developed in apatient after receiving one infusion of CARTmeso cells on Schedule 2.FIG. 6B depicts FDG PET/Ct imaging of the patient. FIG. 6C shows changesin the maximum standardized uptake value (SUVmax) and the meanvolumetric product (MVPmean) for each disease site. FIG. 6D shows theanalysis of ascitic fluid on days +3 and +15 after beginning therapy.

FIG. 7A shows the in vivo persistence of CARTmeso cells. FIG. 7B showsthe analysis of peripheral blood samples from the two patients. FIG. 7Cshows the analysis of ascite and tumor samples from two patients.

FIG. 8 shows cytokine and chemokines in the serum after CARTmeso cellinfusion.

FIG. 9A and FIG. 9B show CARTmeso cell induction of anti-tumorantibodies. Sera was obtained from the MPM patient (FIG. 9A) and thepancreatic cancer patient (FIG. 9B).

FIG. 10 shows tumor growth in NSG mice injected with EMMESO tumor cells.After tumors grew to ˜200 mm³ in size, mesoCAR T cells were injected viatail vein and measured for 39 days post injection.

FIG. 11A and FIG. 11B show the expression of mesoCAR by flow cytometryanalysis at the time of injection (FIG. 11A) or after 40 days at thetime of harvest from xenograft tumors (FIG. 11B).

FIG. 12 shows the functional capacity of mesoCAR T cells with regard toin vitro killing when isolated from the flank of NSG mice after 39 days,or cryo preserved after transduction.

FIG. 13 shows the expression of inhibitor enzymes DGK and SHP1 in TILsisolated from EMESO flank tumor compared to overnight-rested TILs.

FIG. 14A, FIG. 14B, FIG. 14C, FIG. 14D, FIG. 14E, and FIG. 14F show theeffect of treatment with inhibitors (anti-PDL1, DGK inhibitor, and SSG)of inhibitory mechanisms that downregulate mesoCART function on tumorcell killing (FIG. 14A, FIG. 14C and FIG. 14E) and IFNgamma cytokinesecretion (FIG. 14B, FIG. 14D, and FIG. 14F).

FIG. 15A, FIG. 15B, FIG. 15C, and FIG. 15D show cytokine secretion froma small panel of human CART-MSLN after stimulation with various tumorcell lines. FIG. 15A shows IFNgamma secretion. FIG. 15B shows TNF. FIG.15C shows IL-2. FIG. 15D shows IL-4.

FIG. 16A and FIG. 16B show the results of the killing assay ofCART-MSLN-5, CART-MSLN-11, CART-MSLN17, and murine CART-MSLN-SS1 againstOvcar3 (FIG. 16A) and U87 mg (FIG. 16B) tumor cells.

FIG. 17A and FIG. 17B show the results of the killing assay of the panelof CART-MSLN against Ovcar3 tumor cells. FIG. 17A depicts killing assayresults for M1-M12, SS1, and UTD. FIG. 17B depicts killing assay resultsfor M13-M24, muCAR19, and UTD.

FIG. 18 shows the anti-tumor activity of a first set of CART-MSLN(including M5, M11, M17, and M21) in the Ovcar8 xenograft model.

FIG. 19 shows the anti-tumor activity of a second set of CART-MSLN(including M12, M14, M16, and M23) in the Ovcar8 xenograft model.

FIG. 20A, FIG. 20B, and FIG. 20C depict the loss of fucntionality ofmesoCAR T cells in the tumor microenvironment (TILs) over time comparedto fresh or thawed mesoCAR T cells. FIG. 20A shows a cytotoxicity assay;FIG. 20B shows an IFNγ release assay; and FIG. 20C shows a western blotanalysis of ERK signaling (via phosphorylation).

FIG. 21 depicts the effect of deletion of DGK on cytotoxicity of mesoCART cells. Percent target cell killing is assessed at differenteffector:target ratios.

FIG. 22 depicts the effect of deletion of DGK on IFNγ production andrelease from mesoCAR T cells. Concentration of IFNγ is assessed atdifferent effector:target ratios.

FIG. 23 depicts the effect of deletion of DGK on ERK signaling, or Tcell activation, mesoCAR T cells. B: albumin, M: mesothelin, 3/28:CD3/CD28 stimulated cells.

FIG. 24 depicts the effect of deletion of DGK on TGFβ sensitivity ofmesoCAR T cells with regard to cytotoxic activity.

FIG. 25A and FIG. 25B depict the effect of deletion of DGK ontherapeutic efficacy of mesoCAR T cells in a tumor mouse model. FIG. 25Ashows the effect on anti-tumor activity by monitoring tumor volume overtime. FIG. 25B shows the persistance and proliferation of tumorinfiltrating cells.

FIG. 26A, FIG. 26B, FIG. 26C, FIG. 26D, FIG. 26E, and FIG. 26F show thecytokine production and cytotoxic mediator release in CAR-expressing Tcells with reduced levels of Ikaros. FIG. 26A shows Ikaros expression inwild-type and Ikzf1+/− CAR T cells as measured by flow cytometry (leftpanel) and western blot (right panel). Following stimulation withmesothelin-coated beads, PMA/Ionomycin (PMA/I), or BSA-coated beads(control), the percentage of cells producing IFN-γ (FIG. 26B), TNF-α(FIG. 26C), and IL-2 (FIG. 26D), the cytotoxic mediator granzyme B (FIG.26E), and CD107a expression (FIG. 26F) was determined.

FIG. 27A, FIG. 27B, and FIG. 27C show cytokine production and cytotoxicmediator release in CAR-expressing T cells with a dominant negativeallele of Ikaros (IkDN). Following stimulation with mesothelin-coatedbeads, PMA/Ionomycin (PMA/I), or BSA-coated beads (control), thepercentage of cells producing IFN-γ (FIG. 27A), IL-2 (FIG. 27B), andCD107a expression (FIG. 27C) was determined.

FIG. 28A, FIG. 28B, FIG. 28C, FIG. 28D, and FIG. 28E show that thedepletion of Ikaros did not augment activation and signaling of CAR Tcells following antigen stimulation. The levels of CD69 (FIG. 28A), CD25(FIG. 28B), and 4-1BB (FIG. 28C) was determined by flow cytometry at theindicated time points in Ikzf1+/− CAR T cells. In FIG. 28D, the RAS/ERKsignaling pathways were examined in wild-type (WT) and Ikaros dominantnegative cells (IkDN) after TCR stimulation with CD3/CD28 antibodies.The levels of phosphorylated TCR signaling proteins such asphosphorylated PLCγ, phosphorylated Lck, phosphorylated JNK,phosphorylated Akt, phosphorylated ERK, phosphorylated IKKα, and IκBαwere assessed by western blot. In FIG. 28E, WT and IkDN cells transducedwith mesoCAR were stimulated with BSA or mesothelin-coated beads, anddownstream signaling pathways were examined by western blot by assessingthe levels of phosphorylated ERK and phosphorylated PLCγ.

FIG. 29A, FIG. 29B, FIG. 29C, FIG. 29D, and FIG. 29E show that thereduction of Ikaros in CAR T cells augments the response against targetcells AE17 or mesothelin-expressing AE17 (AE17 meso) in vitro. FIG. 29Adepicts IFNγ production in WT and Ikzf1+/− meso CART cells at theindicated effector:target cell ratios. Cytolysis of meso CAR-expressingWT and Ikzf1+/− (FIG. 29B) and IkDN (FIG. 29C) was measured at theindicated effector:target cell ratios. IFNγ production (FIG. 29D) andcytolysis (FIG. 29E) of WT and Ikzf1+/− transduced with FAP-CAR wasmeasured at the indicated effector:target cell ratios, where the targetcells were FAP-expressing 3T3 cells.

FIG. 30A, FIG. 30B, and FIG. 30C show the efficacy of CART cells withdepletion of Ikaros against established tumors in vivo. CAR T cells wereadministered to mice bearing established mesothelin-expressing AE17tumors. Tumor volume was measured after administration withmesoCAR-expressing WT and Ikzf1+/− (FIG. 30A) or IkDN (FIG. 30B). Tumorvolume was measured after administration of FAP-CAR-expressing WT andIkzf1+1-(FIG. 30C).

FIG. 31A, FIG. 31B, FIG. 31C, FIG. 31D, FIG. 31E, and FIG. 31F show theincreased persistence and resistance of Ikzf1+/− CAR T cells in theimmunosuppressive tumor microenvironment compared to WT CAR T cells. Thepercentage of CAR-expressing WT or Ikzf1+/− cells (GFP positive) weredetected by flow cytometry from harvested from the spleen (FIG. 31A) andthe tumors (FIG. 31B). The functional capacity of the CAR T cellsharvested 3 days after infusion from the spleen or tumors was assessedby measuring IFNγ production after stimulation with CD3/CD28 antibodies(FIG. 31C) or PMA/Ionomycin (PMA/I) (FIG. 31D). Regulatory T cells(CD4+FoxP3+ expression) and macrophages (CD206 expression) were assessedby measuring the expression of Treg (FIG. 31E) or macrophage markers(FIG. 31F) on CAR T cells harvested 9 days after infusion from thespleen or tumors.

FIG. 32A and FIG. 32B show that T cells with reduced Ikaros levels areless sensitive to soluble inhibitory factors TGFβ and adenosine.MesoCAR-expressing WT, Ikzf1+/−, and IkDN cells were tested for theirability to produce IFNγ (FIG. 32A) and cytotoxicity (FIG. 32B) inresponse to TGF-β or adenosine.

FIG. 33A and FIG. 33B are graphs showing an increase in titers toinfluenza vaccine strains as compared to placebo. In FIG. 33A, theincrease above baseline in influenza geometric mean titers to each ofthe 3 influenza vaccine strains (H1N1 A/California/07/2009, H3N2A/Victoria/210/2009, B/Brisbane/60/2008) relative to the increase in theplacebo cohort 4 weeks after vaccination is shown for each of the RAD001dosing cohorts in the intention to treat population. The bold black lineindicates the 1.2 fold increase in titers relative to placebo that isrequired to be met for 2 out of 3 influenza vaccine strains to meet theprimary endpoint of the study. The star “*” indicates that the increasein GMT titer relative to placebo exceeds 1 with posterior probability ofat least 80%. FIG. 33B is a graph of the same data as in FIG. 33A forthe subset of subjects with baseline influenza titers <=1:40.

FIG. 34 shows a scatter plot of RAD001 concentration versus foldincrease in geometric mean titer to each influenza vaccine strain 4weeks after vaccination. RAD001 concentrations (1 hour post dose) weremeasured after subjects had been dosed for 4 weeks. All subjects who hadpharmacokinetic measurements were included in the analysis set. The foldincrease in geometric mean titers at 4 weeks post vaccination relativeto baseline is shown on the y axis.

FIG. 35 is a graphic representation showing increase in titers toheterologous influenza strains as compared to placebo. The increaseabove baseline in influenza geometric mean titers to 2 heterologousinfluenza strains (A/H1N1 strain A/New Jersey/8/76 and A/H3N2 strainA/Victoria/361/11) not contained in the influenza vaccine relative tothe increase in the placebo cohort 4 weeks after vaccination is shownfor each of the RAD001 dosing cohorts in the intention to treatpopulation. * indicates increase in titer relative to placebo exceeds 1with a posterior probability of at least 80%.

FIG. 36A and FIG. 36B are graphic representations of IgG and IgM levelsbefore and after influenza vaccination. Levels ofanti-A/H1N1/California/07/2009 influenza IgG (FIG. 36A) and IgM (FIG.36B) were measured in serum obtained from subjects before and 4 weekspost influenza vaccination. No significant difference in the change frombaseline to 4 weeks post vaccination in anti-H1N1 influenza IgG and IgMlevels were detected between the RAD001 and placebo cohorts (all pvalues >0.05 by Kruskal-Wallis rank sum test).

FIG. 37A, FIG. 37B, and FIG. 37C are graphic representations of thedecrease in percent of PD-1-positive CD4 and CD8 and increase inPD-1-negative CD4 T cells after RAD001 treatment. The percent ofPD-1-positive CD4, CD8 and PD-1-negative CD4 T cells was determined byFACS analysis of PBMC samples at baseline, after 6 weeks of study drugtreatment (Week 6) and 6 weeks after study drug discontinuation and 4weeks after influenza vaccination (Week 12). FIG. 37A shows there was asignificant decrease (−37.1-−28.5%) in PD-1-positive CD4 T cells at week12 in cohorts receiving RAD001 at dose levels 0.5 mg/Day (n=25), 5mg/Week (n=29) and 20 mg/Week (n=30) as compared to the placebo cohort(n=25) with p=0.002 (0.02), p=0.003 (q=0.03), and p=0.01 (q=0.05)respectively. FIG. 37B shows there was a significant decrease(−43.3-−38.5%) in PD-1-positive CD8 T cells at week 12 in cohortsreceiving RAD001 (n=109) at dose levels 0.5 mg/Day (n=25), 5 mg/Week(n=29) and 20 mg/Week (n=30) as compared to the placebo cohort (n=25)with p=0.01 (0.05), p=0.007 (q=0.04), and p=0.01 (q=0.05) respectively.FIG. 37C shows was a significant increase (3.0-4.9%) in PD-1-negativeCD4 T cells at week 12 in cohorts receiving RAD001 (n=109) at doselevels 0.5 mg/Day (n=25), 5 mg/Week (n=29) and 20 mg/Week (n=30) ascompared to the placebo cohort (n=25) with p=0.0007 (0.02), p=0.03(q=0.07), and p=0.03 (q=0.08) respectively.

FIG. 38A and FIG. 38B are graphic representations of the decrease inpercent of PD-1-positive CD4 and CD8 and increase in PD-1-negative CD4 Tcells after RAD001 treatment adjusted for differences in baseline PD-1expression. The percent of PD-1-positive CD4, CD8 and PD-1-negative CD4T cells was determined by FACS analysis of PBMC samples at baseline,after 6 weeks of study drug treatment (Week 6) and 6 weeks after studydrug discontinuation and 4 weeks after influenza vaccination (Week 12).FIG. 38A shows a significant decrease of 30.2% in PD-1+CD4 T cells atweek 6 in the pooled RAD cohort (n=84) compared to placebo cohort (n=25)with p=0.03 (q=0.13). The decrease in PD-1-positive CD4 T cells at week12 in the pooled RAD as compared to the placebo cohort is 32.7% withp=0.05 (q=0.19). FIG. 38B shows a significant decrease of 37.4% inPD-1-positive CD8 T cells at week 6 in the pooled RAD001 cohort (n=84)compared to placebo cohort (n=25) with p=0.008 (q=0.07). The decrease inPD-1-positive CD8 T cells at week 12 in the pooled RAD001 as compared tothe placebo cohort is 41.4% with p=0.066 (q=0.21). FIGS. 38A and 38Brepresent the data in FIGS. 37A, 37B, and 37C but with the differentRAD001 dosage groups of FIGS. 37A, 37B, and 37C pooled into the singleRAD001-treated group in FIGS. 38A and 38B.

FIG. 39 depicts increases in exercise and energy in elderly subjects inresponse to RAD001.

FIG. 40A and FIG. 40B depict the predicted effect of RAD001 on P70 S6Kactivity in cells. FIG. 40A depicts P70 S6 kinase inhibition with higherdoses of weekly and daily RAD001; FIG. 40B depicts P70 S6 kinaseinhibition with lower doses of weekly RAD001.

FIG. 41A, FIG. 41B, and FIG. 41C are Biacore T200 SPR sensograms for thescFvs SS1 (FIG. 41A), M5 (FIG. 41B), and M11 (FIG. 41C).

FIG. 42A, FIG. 42B, and FIG. 42C are epitope binning SPR sensograms forthe anti-human mesothelin scFvs in comparison to the murine SS1 scFv.Competitive binding was observed for scFvs M12, M14, M16, M17, M21, andM23 (FIG. 42A). ScFv M5 (FIG. 42B) and M11 (FIG. 42C) bind to adifferent epitope than SS1.

FIG. 43 is a graph depicting tumor growth after various mesothelin CAR Ttreatments in the OVCAR8 tumor model. Mean tumor volume+/−SEM to day 62post tumor implantation. T cells were administered on days 14 and 19.Small circles: mice treated with 100 ul of PBS via the lateral tailvein; black squares: mice treated with Isotype control T cells; graytriangles: mice treated with one dose of SS1 CART cells; invertedtriangles: mice treated with a double dose of SS1 CART cells; diamonds:mice treated with a single dose of M5 CAR T cells; large circles: micetreated with a double dose of M5 CAR T cells; gray squares: mice treatedwith a single dose of M11 CAR T cells; and black triangles: mice treatedwith a double dose of M11 CAR T cells.

FIG. 44 is a schematic representation of the human mesothelin peptidecoverage in hydrogen deuterium exchange mass spectrometry analysis. Eachblack bar represents a peptide.

FIG. 45A and FIG. 45B are graphic representations showing the differencein deuterium uptake of human mesothelin when in complex with SS1 (blackbars) and M5 (grey bars). The difference in deuterium uptake uponantibody binding (represented on the y-axis) is depicted for eachpeptide fragment detected (represented on the x-axis), with peptides atamino acids 297-464 in FIG. 45A and peptides at amino acids 458-586 inFIG. 45B. All differences are relative to the deuterium uptake ofunbound mesothelin (control). * denote regions of statisticalsignificance using the Tukey test for peptides with a difference lessthan 0.75 Da.

FIG. 46 is a schematic representation showing the primary sequence ofantigen human mesothelin (amino acids 296-588) and the regions protectedby SS1 and M5. The black bars designate the amino acids protected whencomplexed with SS1 (amino acids 314-315, 317-318, 346-349, and 369-375).The grey bars designate the amino acids protected when complexed with M5(amino acids 485-490, 498-507, 532-537, and 545-572).

FIG. 47A, FIG. 47B, FIG. 47C, FIG. 47D, and FIG. 47E depict a genericmap showing different configurations of constructs encoding a CAR with ashRNA for coexpression of the CAR and an shRNA. FIGS. 47A-47D show thevarious configurations on a single vector, e.g., where the U6 regulatedshRNA is upstream or downstream of the EF1 alpha regulated CAR encodingelements. In the exemplary constructs depicted in FIG. 47A and FIG. 47B,the transcription occurs through the U6 and EF1 alpha promoters in thesame direction. In the exemplary constructs depicted in FIG. 47C andFIG. 47D, the transcription occurs through the U6 and EF1 alphapromoters in different directions. In FIG. 47E, the shRNA (andcorresponding U6 promoter) is on a first vector, and the CAR (andcorresponding EF1 alpha promoter) is on a second vector.

FIG. 48 depicts the structures of two exemplary RCAR configurations. Theantigen binding members comprise an antigen binding domain, atransmembrane domain, and a switch domain. The intracellular bindingmembers comprise a switch domain, a co-stimulatory signaling domain anda primary signaling domain. The two configurations demonstrate that thefirst and second switch domains described herein can be in differentorientations with respect to the antigen binding member and theintracellular binding member. Other RCAR configurations are furtherdescribed herein.

DETAILED DESCRIPTION Definitions

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which the invention pertains.

The term “a” and “an” refers to one or to more than one (i.e., to atleast one) of the grammatical object of the article. By way of example,“an element” means one element or more than one element.

The term “about” when referring to a measurable value such as an amount,a temporal duration, and the like, is meant to encompass variations of±20% or in some instances ±10%, or in some instances ±5%, or in someinstances ±1%, or in some instances ±0.1% from the specified value, assuch variations are appropriate to perform the disclosed methods.

The term “Chimeric Antigen Receptor” or alternatively a “CAR” refers toa set of polypeptides, typically two in the simplest embodiments, whichwhen in an immune effector cell, provides the cell with specificity fora target cell, typically a cancer cell, and with intracellular signalgeneration. In some embodiments, a CAR comprises at least anextracellular antigen binding domain, a transmembrane domain and acytoplasmic signaling domain (also referred to herein as “anintracellular signaling domain”) comprising a functional signalingdomain derived from a stimulatory molecule and/or costimulatory moleculeas defined below. In some aspects, the set of polypeptides arecontiguous with each other. In some embodiments, the set of polypeptidesinclude a dimerization switch that, upon the presence of a dimerizationmolecule, can couple the polypeptides to one another, e.g., can couplean antigen binding domain to an intracellular signaling domain. In oneaspect, the stimulatory molecule is the zeta chain associated with the Tcell receptor complex. In one aspect, the cytoplasmic signaling domainfurther comprises one or more functional signaling domains derived fromat least one costimulatory molecule as defined below. In one aspect, thecostimulatory molecule is chosen from the costimulatory moleculesdescribed herein, e.g., 4-1BB (i.e., CD137), CD27 and/or CD28. In oneaspect, the CAR comprises a chimeric fusion protein comprising anextracellular antigen binding domain, a transmembrane domain and anintracellular signaling domain comprising a functional signaling domainderived from a stimulatory molecule. In one aspect, the CAR comprises achimeric fusion protein comprising an extracellular antigen bindingdomain, a transmembrane domain and an intracellular signaling domaincomprising a functional signaling domain derived from a costimulatorymolecule and a functional signaling domain derived from a stimulatorymolecule. In one aspect, the CAR comprises a chimeric fusion proteincomprising an extracellular antigen binding domain, a transmembranedomain and an intracellular signaling domain comprising two functionalsignaling domains derived from one or more costimulatory molecule(s) anda functional signaling domain derived from a stimulatory molecule. Inone aspect, the CAR comprises a chimeric fusion protein comprising anextracellular antigen binding domain, a transmembrane domain and anintracellular signaling domain comprising at least two functionalsignaling domains derived from one or more costimulatory molecule(s) anda functional signaling domain derived from a stimulatory molecule. Inone aspect the CAR comprises an optional leader sequence at theamino-terminus (N-ter) of the CAR fusion protein. In one aspect, the CARfurther comprises a leader sequence at the N-terminus of theextracellular antigen binding domain, wherein the leader sequence isoptionally cleaved from the antigen binding domain (e.g., a scFv) duringcellular processing and localization of the CAR to the cellularmembrane.

The term “signaling domain” refers to the functional portion of aprotein which acts by transmitting information within the cell toregulate cellular activity via defined signaling pathways by generatingsecond messengers or functioning as effectors by responding to suchmessengers.

As used herein, the term “mesothelin” refers to the 40-kDa protein,mesothelin, which is anchored at the cell membrane by aglycosylphosphatidyl inositol (GPI) linkage and an amino-terminal 31-kDashed fragment, called megkaryocyte potentiating factor (MPF). Bothfragments contain N-glycosylation sites. The term also refers to asoluble splice variant of the 40-kDa carboxyl-terminal fragment alsocalled “soluble mesothelin/MPF-related”. Preferably, the term refers toa human mesothelin of GenBank accession number AAH03512.1, and naturallycleaved portions thereof, e.g., as expressed on a cell membrane, e.g., acancer cell membrane.

The term “antibody” as used herein, refers to a protein, or polypeptidesequence derived from an immunoglobulin molecule which specificallybinds with an antigen. Antibodies can be polyclonal or monoclonal,multiple or single chain, or intact immunoglobulins, and may be derivedfrom natural sources or from recombinant sources. Antibodies can betetramers of immunoglobulin molecules.

The term “antibody fragment” refers to at least one portion of anantibody, that retains the ability to specifically interact with (e.g.,by binding, steric hinderance, stabilizing/destabilizing, spatialdistribution) an epitope of an antigen. Examples of antibody fragmentsinclude, but are not limited to, Fab, Fab′, F(ab′)2, Fv fragments, scFvantibody fragments, disulfide-linked Fvs (sdFv), a Fd fragmentconsisting of the VH and CH1 domains, linear antibodies, single domainantibodies such as sdAb (either VL or VH), camelid VHH domains,multi-specific antibodies formed from antibody fragments such as abivalent fragment comprising two Fab fragments linked by a disulfidebrudge at the hinge region, and an isolated CDR or other epitope bindingfragments of an antibody. An antigen binding fragment can also beincorporated into single domain antibodies, maxibodies, minibodies,nanobodies, intrabodies, diabodies, triabodies, tetrabodies, v-NAR andbis-scFv (see, e.g., Hollinger and Hudson, Nature Biotechnology23:1126-1136, 2005). Antigen binding fragments can also be grafted intoscaffolds based on polypeptides such as a fibronectin type III (Fn3)(seeU.S. Pat. No. 6,703,199, which describes fibronectin polypeptideminibodies).

The term “scFv” refers to a fusion protein comprising at least oneantibody fragment comprising a variable region of a light chain and atleast one antibody fragment comprising a variable region of a heavychain, wherein the light and heavy chain variable regions arecontiguously linked, e.g., via a synthetic linker, e.g., a shortflexible polypeptide linker, and capable of being expressed as a singlechain polypeptide, and wherein the scFv retains the specificity of theintact antibody from which it is derived. Unless specified, as usedherein an scFv may have the VL and VH variable regions in either order,e.g., with respect to the N-terminal and C-terminal ends of thepolypeptide, the scFv may comprise VL-linker-VH or may compriseVH-linker-VL.

The portion of the CAR of the invention comprising an antibody orantibody fragment thereof may exist in a variety of forms where theantigen binding domain is expressed as part of a contiguous polypeptidechain including, for example, a single domain antibody fragment (sdAb),a single chain antibody (scFv) a humanized antibody or bispecificantibody (Harlow et al., 1999, In: Using Antibodies: A LaboratoryManual, Cold Spring Harbor Laboratory Press, NY; Harlow et al., 1989,In: Antibodies: A Laboratory Manual, Cold Spring Harbor, N.Y.; Houstonet al., 1988, Proc. Natl. Acad. Sci. USA 85:5879-5883; Bird et al.,1988, Science 242:423-426). In one aspect, the antigen binding domain ofa CAR composition of the invention comprises an antibody fragment. In afurther aspect, the CAR comprises an antibody fragment that comprises ascFv.

The term “antibody heavy chain” refers to the larger of the two types ofpolypeptide chains present in antibody molecules in their naturallyoccurring conformations, and which normally determines the class towhich the antibody belongs.

The term “antibody light chain” refers to the smaller of the two typesof polypeptide chains present in antibody molecules in their naturallyoccurring conformations. Kappa (κ) and lambda (λ) light chains refer tothe two major antibody light chain isotypes.

The term “recombinant antibody” refers to an antibody which is generatedusing recombinant DNA technology, such as, for example, an antibodyexpressed by a bacteriophage or yeast expression system. The term shouldalso be construed to mean an antibody which has been generated by thesynthesis of a DNA molecule encoding the antibody and which DNA moleculeexpresses an antibody protein, or an amino acid sequence specifying theantibody, wherein the DNA or amino acid sequence has been obtained usingrecombinant DNA or amino acid sequence technology which is available andwell known in the art.

The term “antigen” or “Ag” refers to a molecule that provokes an immuneresponse. This immune response may involve either antibody production,or the activation of specific immunologically-competent cells, or both.The skilled artisan will understand that any macromolecule, includingvirtually all proteins or peptides, can serve as an antigen.Furthermore, antigens can be derived from recombinant or genomic DNA. Askilled artisan will understand that any DNA, which comprises anucleotide sequences or a partial nucleotide sequence encoding a proteinthat elicits an immune response therefore encodes an “antigen” as thatterm is used herein. Furthermore, one skilled in the art will understandthat an antigen need not be encoded solely by a full length nucleotidesequence of a gene. It is readily apparent that the present inventionincludes, but is not limited to, the use of partial nucleotide sequencesof more than one gene and that these nucleotide sequences are arrangedin various combinations to encode polypeptides that elicit the desiredimmune response. Moreover, a skilled artisan will understand that anantigen need not be encoded by a “gene” at all. It is readily apparentthat an antigen can be generated synthesized or can be derived from abiological sample, or might be macromolecule besides a polypeptide. Sucha biological sample can include, but is not limited to a tissue sample,a tumor sample, a cell or a fluid with other biological components.

The term “compete” refers to the ability of an antigen binding domain,e.g., an antibody or fragment thereof, to interfere with bindingdirectly or indirectly of another antigen binding domain, e.g., anantigen binding domain provided herein, e.g., an antibody or fragmentthereof provided herein, to the target, e.g., mesothelin. The extent towhich an antigen binding domain, e.g., an antibody or fragment thereof,is able to interfere with the binding of another antigen binding domain,e.g., an antibody or fragment thereof, to the target, and thereforewhether it can be said to compete, can be determined using a competitionbinding assay. In some embodiments, a competition binding assay is aquantitative competition assay. For example, one particularly suitablequantitative competition assay uses a surface plasmon resonance(SPR)-based approach to measure binding, e.g., competition, between oneantibody or fragment thereof and another antibody or fragment thereoffor binding to an immobilized target. An exemplary SPR-based competitionassay is described in Example 2 herein. Another suitable quantitativecompetition assay uses a FACS-based approach to measure competitionbetween a labelled (e.g., His tagged, biotinylated or radioactivelylabeled, among others) antibody or fragment thereof and another antibodyor fragment thereof for binding to the target.

The term “anti-cancer effect” refers to a biological effect which can bemanifested by various means, including but not limited to, e.g., adecrease in tumor volume, a decrease in the number of cancer cells, adecrease in the number of metastases, an increase in life expectancy,decrease in cancer cell proliferation, decrease in cancer cell survival,or amelioration of various physiological symptoms associated with thecancerous condition. An “anti-cancer effect” can also be manifested bythe ability of the peptides, polynucleotides, cells and antibodies inprevention of the occurrence of cancer in the first place. The term“anti-tumor effect” refers to a biological effect which can bemanifested by various means, including but not limited to, e.g., adecrease in tumor volume, a decrease in the number of tumor cells, adecrease in tumor cell proliferation, or a decrease in tumor cellsurvival.

The term “autologous” refers to any material derived from the sameindividual to whom it is later to be re-introduced into the individual.

The term “allogeneic” refers to any material derived from a differentanimal of the same species as the individual to whom the material isintroduced. Two or more individuals are said to be allogeneic to oneanother when the genes at one or more loci are not identical. In someaspects, allogeneic material from individuals of the same species may besufficiently unlike genetically to interact antigenically.

The term “xenogeneic” refers to a graft derived from an animal of adifferent species.

The term “cancer” refers to a disease characterized by the uncontrolledgrowth of aberrant cells. Cancer cells can spread locally or through thebloodstream and lymphatic system to other parts of the body. Examples ofvarious cancers are described herein and include, but are not limitedto, mesothelioma, breast cancer, prostate cancer, ovarian cancer,cervical cancer, skin cancer, pancreatic cancer, colorectal cancer,renal cancer, liver cancer, brain cancer, lymphoma, leukemia, lungcancer and the like.

The phrase “disease associated with expression of mesothelin” includes,but is not limited to, a disease associated with expression ofmesothelin or condition associated with cells which express mesothelinincluding, e.g., proliferative diseases such as a cancer or malignancyor a precancerous condition such as a mesothelial hyperplasia; or anoncancer related indication associated with cells which expressmesothelin. Examples of various cancers that express mesothelin includebut are not limited to, mesothelioma, lung cancer, ovarian cancer,pancreatic cancer, and the like.

The term “conservative sequence modifications” refers to amino acidmodifications that do not significantly affect or alter the bindingcharacteristics of the antibody or antibody fragment containing theamino acid sequence. Such conservative modifications include amino acidsubstitutions, additions and deletions. Modifications can be introducedinto an antibody or antibody fragment of the invention by standardtechniques known in the art, such as site-directed mutagenesis andPCR-mediated mutagenesis. Conservative amino acid substitutions are onesin which the amino acid residue is replaced with an amino acid residuehaving a similar side chain. Families of amino acid residues havingsimilar side chains have been defined in the art. These families includeamino acids with basic side chains (e.g., lysine, arginine, histidine),acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polarside chains (e.g., glycine, asparagine, glutamine, serine, threonine,tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine,valine, leucine, isoleucine, proline, phenylalanine, methionine),beta-branched side chains (e.g., threonine, valine, isoleucine) andaromatic side chains (e.g., tyrosine, phenylalanine, tryptophan,histidine). Thus, one or more amino acid residues within a CAR of theinvention can be replaced with other amino acid residues from the sameside chain family and the altered CAR can be tested, e.g., for theability to bind mesothelin using the functional assays described herein.

The term “stimulation,” refers to a primary response induced by bindingof a stimulatory molecule (e.g., a TCR/CD3 complex or CAR) with itscognate ligand (or tumor antigen in the case of a CAR) thereby mediatinga signal transduction event, such as, but not limited to, signaltransduction via the TCR/CD3 complex or signal transduction via theappropriate NK receptor or signaling domains of the CAR. Stimulation canmediate altered expression of certain molecules.

The term “stimulatory molecule,” refers to a molecule expressed by animmune cell (e.g., T cell, NK cell, B cell) that provides thecytoplasmic signaling sequence(s) that regulate activation of the immunecell in a stimulatory way for at least some aspect of the immune cellsignaling pathway. In one aspect, the signal is a primary signal that isinitiated by, for instance, binding of a TCR/CD3 complex with an MHCmolecule loaded with peptide, and which leads to mediation of a T cellresponse, including, but not limited to, proliferation, activation,differentiation, and the like. A primary cytoplasmic signaling sequence(also referred to as a “primary signaling domain”) that acts in astimulatory manner may contain a signaling motif which is known asimmunoreceptor tyrosine-based activation motif or ITAM. Examples of anITAM containing cytoplasmic signaling sequence that is of particular usein the invention includes, but is not limited to, those derived from CD3zeta, common FcR gamma (FCER1G), Fc gamma RIIa, FcR beta (Fc EpsilonRib), CD3 gamma, CD3 delta, CD3 epsilon, CD79a, CD79b, DAP10, and DAP12.In a specific CAR of the invention, the intracellular signaling domainin any one or more CARS of the invention comprises an intracellularsignaling sequence, e.g., a primary signaling sequence of CD3-zeta. In aspecific CAR of the invention, the primary signaling sequence ofCD3-zeta is the sequence provided as SEQ ID NO:9, or the equivalentresidues from a non-human species, e.g., mouse, rodent, monkey, ape andthe like. In a specific CAR of the invention, the primary signalingsequence of CD3-zeta is the sequence as provided in SEQ ID NO:10, or theequivalent residues from a non-human species, e.g., mouse, rodent,monkey, ape and the like.

The term “antigen presenting cell” or “APC” refers to an immune systemcell such as an accessory cell (e.g., a B-cell, a dendritic cell, andthe like) that displays a foreign antigen complexed with majorhistocompatibility complexes (MHC's) on its surface. T-cells mayrecognize these complexes using their T-cell receptors (TCRs). APCsprocess antigens and present them to T-cells.

An “intracellular signaling domain,” as the term is used herein, refersto an intracellular portion of a molecule. The intracellular signalingdomain generates a signal that promotes an immune effector function ofthe CAR containing cell, e.g., a CART cell. Examples of immune effectorfunction, e.g., in a CART cell, include cytolytic activity and helperactivity, including the secretion of cytokines.

In an embodiment, the intracellular signaling domain can comprise aprimary intracellular signaling domain. Exemplary primary intracellularsignaling domains include those derived from the molecules responsiblefor primary stimulation, or antigen dependent simulation. In anembodiment, the intracellular signaling domain can comprise acostimulatory intracellular domain. Exemplary costimulatoryintracellular signaling domains include those derived from moleculesresponsible for costimulatory signals, or antigen independentstimulation. For example, in the case of a CART, a primary intracellularsignaling domain can comprise a cytoplasmic sequence of a T cellreceptor, and a costimulatory intracellular signaling domain cancomprise cytoplasmic sequence from co-receptor or costimulatorymolecule.

A primary intracellular signaling domain can comprise a signaling motifwhich is known as an immunoreceptor tyrosine-based activation motif orITAM. Examples of ITAM containing primary cytoplasmic signalingsequences include, but are not limited to, those derived from CD3 zeta,common FcR gamma (FCER1G), Fc gamma RIIa, FcR beta (Fc Epsilon R1b), CD3gamma, CD3 delta, CD3 epsilon, CD79a, CD79b, DAP10, and DAP12.

The term “zeta” or alternatively “zeta chain”, “CD3-zeta” or “TCR-zeta”is defined as the protein provided as GenBan Acc. No. BAG36664.1, or theequivalent residues from a non-human species, e.g., mouse, rodent,monkey, ape and the like, and a “zeta stimulatory domain” oralternatively a “CD3-zeta stimulatory domain” or a “TCR-zeta stimulatorydomain” is defined as the amino acid residues from the cytoplasmicdomain of the zeta chain, or functional derivatives thereof, that aresufficient to functionally transmit an initial signal necessary for Tcell activation. In one aspect the cytoplasmic domain of zeta comprisesresidues 52 through 164 of GenBank Acc. No. BAG36664.1 or the equivalentresidues from a non-human species, e.g., mouse, rodent, monkey, ape andthe like, that are functional orthologs thereof. In one aspect, the“zeta stimulatory domain” or a “CD3-zeta stimulatory domain” is thesequence provided as SEQ ID NO:9. In one aspect, the “zeta stimulatorydomain” or a “CD3-zeta stimulatory domain” is the sequence provided asSEQ ID NO:10.

The term a “costimulatory molecule” refers to a cognate binding partneron a T cell that specifically binds with a costimulatory ligand, therebymediating a costimulatory response by the T cell, such as, but notlimited to, proliferation. Costimulatory molecules are cell surfacemolecules other than antigen receptors or their ligands that arecontribute to an efficient immune response. Costimulatory moleculesinclude, but are not limited to an MHC class I molecule, BTLA and a Tollligand receptor, as well as OX40, CD27, CD28, CD5, ICAM-1, LFA-1(CD11a/CD18), ICOS (CD278), and 4-1BB (CD137). Further examples of suchcostimulatory molecules include CD5, ICAM-1, GITR, BAFFR, HVEM (LIGHTR),SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD160, CD19, CD4, CD8alpha,CD8beta, IL2R beta, IL2R gamma, IL7R alpha, ITGA4, VLA1, CD49a, ITGA4,IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL,CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18,LFA-1, ITGB7, NKG2D, NKG2C, TNFR2, TRANCE/RANKL, DNAM1 (CD226), SLAMF4(CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRTAM, Ly9 (CD229), CD160(BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Ly108), SLAM(SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, LAT, GADS,SLP-76, PAG/Cbp, and a ligand that specifically binds with CD83

A costimulatory intracellular signaling domain can be an intracellularportion of a costimulatory molecule. A costimulatory molecule can berepresented in the following protein families: TNF receptor proteins,Immunoglobulin-like proteins, cytokine receptors, integrins, signalinglymphocytic activation molecules (SLAM proteins), and activating NK cellreceptors. Examples of such molecules include CD27, CD28, 4-1BB (CD137),OX40, GITR, CD30, CD40, ICOS, BAFFR, HVEM, ICAM-1, lymphocytefunction-associated antigen-1 (LFA-1), CD2, CD5, CD7, CD287, LIGHT,NKG2C, SLAMF7, NKp80, CD160, B7-H3, and a ligand that specifically bindswith CD83, and the like.

The intracellular signaling domain can comprise the entire intracellularportion, or the entire native intracellular signaling domain, of themolecule from which it is derived, or a functional fragment orderivative thereof.

The term “4-1BB” refers to a member of the TNFR superfamily with anamino acid sequence provided as GenBank Acc. No. AAA62478.2, or theequivalent residues from a non-human species, e.g., mouse, rodent,monkey, ape and the like. In one aspect a “4-1BB costimulatory domain”is defined as amino acid residues 214-255 of GenBank Acc. No.AAA62478.2, or the equivalent residues from a non-human species, e.g.,mouse, rodent, monkey, ape and the like. In one aspect, the “4-1BBcostimulatory domain” is the sequence provided as SEQ ID NO:7 or theequivalent residues from a non-human species, e.g., mouse, rodent,monkey, ape and the like.

An “antigen presenting cell,” as used herein, means an immune systemcell such as an accessory cell (e.g., a B-cell, a dendritic cell, andthe like) that displays foreign antigens complexed with majorhistocompatibility complexes (MHC's) on their surfaces. T-cells mayrecognize these complexes using their T-cell receptors (TCRs). APCsprocess antigens and present them to T-cells.

The term “encoding” refers to the inherent property of specificsequences of nucleotides in a polynucleotide, such as a gene, a cDNA, oran mRNA, to serve as templates for synthesis of other polymers andmacromolecules in biological processes having either a defined sequenceof nucleotides (i.e., rRNA, tRNA and mRNA) or a defined sequence ofamino acids and the biological properties resulting therefrom. Thus, agene, cDNA, or RNA, encodes a protein if transcription and translationof mRNA corresponding to that gene produces the protein in a cell orother biological system. Both the coding strand, the nucleotide sequenceof which is identical to the mRNA sequence and is usually provided insequence listings, and the non-coding strand, used as the template fortranscription of a gene or cDNA, can be referred to as encoding theprotein or other product of that gene or cDNA.

Unless otherwise specified, a “nucleotide sequence encoding an aminoacid sequence” includes all nucleotide sequences that are degenerateversions of each other and that encode the same amino acid sequence. Thephrase nucleotide sequence that encodes a protein or a RNA may alsoinclude introns to the extent that the nucleotide sequence encoding theprotein may in some version contain an intron(s).

The term “effective amount” or “therapeutically effective amount” isused interchangeably herein, and refer to an amount of a compound,formulation, material, or composition, as described herein effective toachieve a particular biological result. The term “endogenous” refers toany material from or produced inside an organism, cell, tissue orsystem.

The term “exogenous” refers to any material introduced from or producedoutside an organism, cell, tissue or system.

The term “expression” refers to the transcription and/or translation ofa particular nucleotide sequence driven by its promoter.

The term “transfer vector” refers to a composition of matter whichcomprises an isolated nucleic acid and which can be used to deliver theisolated nucleic acid to the interior of a cell. Numerous vectors areknown in the art including, but not limited to, linear polynucleotides,polynucleotides associated with ionic or amphiphilic compounds,plasmids, and viruses. Thus, the term “transfer vector” includes anautonomously replicating plasmid or a virus. The term should also beconstrued to further include non-plasmid and non-viral compounds whichfacilitate transfer of nucleic acid into cells, such as, for example, apolylysine compound, liposome, and the like. Examples of viral transfervectors include, but are not limited to, adenoviral vectors,adeno-associated virus vectors, retroviral vectors, lentiviral vectors,and the like.

The term “expression vector” refers to a vector comprising a recombinantpolynucleotide comprising expression control sequences operativelylinked to a nucleotide sequence to be expressed. An expression vectorcomprises sufficient cis-acting elements for expression; other elementsfor expression can be supplied by the host cell or in an in vitroexpression system. Expression vectors include all those known in theart, including cosmids, plasmids (e.g., naked or contained in liposomes)and viruses (e.g., lentiviruses, retroviruses, adenoviruses, andadeno-associated viruses) that incorporate the recombinantpolynucleotide.

The term “lentivirus” refers to a genus of the Retroviridae family.Lentiviruses are unique among the retroviruses in being able to infectnon-dividing cells; they can deliver a significant amount of geneticinformation into the DNA of the host cell, so they are one of the mostefficient methods of a gene delivery vector. HIV, SIV, and FIV are allexamples of lentiviruses. The term “lentiviral vector” refers to avector derived from at least a portion of a lentivirus genome, includingespecially a self-inactivating lentiviral vector as provided in Miloneet al., Mol. Ther. 17(8): 1453-1464 (2009). Other examples of lentivirusvectors that may be used in the clinic include but are not limited to,e.g., the LENTIVECTOR® gene delivery technology from Oxford BioMedica,the LENTIMAX™ vector system from Lentigen and the like. Nonclinicaltypes of lentiviral vectors are also available and would be known to oneskilled in the art.

The term “homologous” or “identity” refers to the subunit sequenceidentity between two polymeric molecules, e.g., between two nucleic acidmolecules, such as, two DNA molecules or two RNA molecules, or betweentwo polypeptide molecules. When a subunit position in both of the twomolecules is occupied by the same monomeric subunit; e.g., if a positionin each of two DNA molecules is occupied by adenine, then they arehomologous or identical at that position. The homology between twosequences is a direct function of the number of matching or homologouspositions; e.g., if half (e.g., five positions in a polymer ten subunitsin length) of the positions in two sequences are homologous, the twosequences are 50% homologous; if 90% of the positions (e.g., 9 of 10),are matched or homologous, the two sequences are 90% homologous.

The term “humanized” refers to those forms of non-human (e.g., murine)antibodies are chimeric immunoglobulins, immunoglobulin chains orfragments thereof (such as Fv, Fab, Fab′, F(ab′)2 or otherantigen-binding subsequences of antibodies) which contain minimalsequence derived from non-human immunoglobulin. For the most part,humanized antibodies and antibody fragments thereof are humanimmunoglobulins (recipient antibody or antibody fragment) in whichresidues from a complementary-determining region (CDR) of the recipientare replaced by residues from a CDR of a non-human species (donorantibody) such as mouse, rat or rabbit having the desired specificity,affinity, and capacity. In some instances, Fv framework region (FR)residues of the human immunoglobulin are replaced by correspondingnon-human residues. Furthermore, a humanized antibody/antibody fragmentcan comprise residues which are found neither in the recipient antibodynor in the imported CDR or framework sequences. These modifications canfurther refine and optimize antibody or antibody fragment performance.In general, the humanized antibody or antibody fragment thereof willcomprise a significant portion of at least one, and typically two,variable domains, in which all or substantially all of the CDR regionscorrespond to those of a non-human immunoglobulin and all orsubstantially all of the FR regions are those of a human immunoglobulinsequence. The humanized antibody or antibody fragment can also compriseat least a portion of an immunoglobulin constant region (Fc), typicallythat of a human immunoglobulin. For further details, see Jones et al.,Nature, 321: 522-525, 1986; Reichmann et al., Nature, 332: 323-329,1988; Presta, Curr. Op. Struct. Biol., 2: 593-596, 1992.

The term “fully human” refers to an immunoglobulin, such as an antibodyor antibody fragment, where the whole molecule is of human origin orconsists of an amino acid sequence identical to a human form of theantibody or immunoglobulin.

The term “isolated” means altered or removed from the natural state. Forexample, a nucleic acid or a peptide naturally present in a livinganimal is not “isolated,” but the same nucleic acid or peptide partiallyor completely separated from the coexisting materials of its naturalstate is “isolated.” An isolated nucleic acid or protein can exist insubstantially purified form, or can exist in a non-native environmentsuch as, for example, a host cell.

In the context of the present invention, the following abbreviations forthe commonly occurring nucleic acid bases are used. “A” refers toadenosine, “C” refers to cytosine, “G” refers to guanosine, “T” refersto thymidine, and “U” refers to uridine.

The term “operably linked” or “transcriptional control” refers tofunctional linkage between a regulatory sequence and a heterologousnucleic acid sequence resulting in expression of the latter. Forexample, a first nucleic acid sequence is operably linked with a secondnucleic acid sequence when the first nucleic acid sequence is placed ina functional relationship with the second nucleic acid sequence. Forinstance, a promoter is operably linked to a coding sequence if thepromoter affects the transcription or expression of the coding sequence.Operably linked DNA sequences can be contiguous with each other and,where necessary to join two protein coding regions, are in the samereading frame.

The term “parenteral” administration of an immunogenic compositionincludes, e.g., subcutaneous (s.c.), intravenous (i.v.), intramuscular(i.m.), or intrasternal injection, intratumoral, or infusion techniques.

The term “nucleic acid” or “polynucleotide” refers to deoxyribonucleicacids (DNA) or ribonucleic acids (RNA) and polymers thereof in eithersingle- or double-stranded form. Unless specifically limited, the termencompasses nucleic acids containing known analogues of naturalnucleotides that have similar binding properties as the referencenucleic acid and are metabolized in a manner similar to naturallyoccurring nucleotides. Unless otherwise indicated, a particular nucleicacid sequence also implicitly encompasses conservatively modifiedvariants thereof (e.g., degenerate codon substitutions), alleles,orthologs, SNPs, and complementary sequences as well as the sequenceexplicitly indicated. Specifically, degenerate codon substitutions maybe achieved by generating sequences in which the third position of oneor more selected (or all) codons is substituted with mixed-base and/ordeoxyinosine residues (Batzer et al., Nucleic Acid Res. 19:5081 (1991);Ohtsuka et al., J. Biol. Chem. 260:2605-2608 (1985); and Rossolini etal., Mol. Cell. Probes 8:91-98 (1994)).

The terms “peptide,” “polypeptide,” and “protein” are usedinterchangeably, and refer to a compound comprised of amino acidresidues covalently linked by peptide bonds. A protein or peptide mustcontain at least two amino acids, and no limitation is placed on themaximum number of amino acids that can comprise a protein's or peptide'ssequence. Polypeptides include any peptide or protein comprising two ormore amino acids joined to each other by peptide bonds. As used herein,the term refers to both short chains, which also commonly are referredto in the art as peptides, oligopeptides and oligomers, for example, andto longer chains, which generally are referred to in the art asproteins, of which there are many types. “Polypeptides” include, forexample, biologically active fragments, substantially homologouspolypeptides, oligopeptides, homodimers, heterodimers, variants ofpolypeptides, modified polypeptides, derivatives, analogs, fusionproteins, among others. A polypeptide includes a natural peptide, arecombinant peptide, a recombinant peptide, or a combination thereof.

The term “promoter” refers to a DNA sequence recognized by the syntheticmachinery of the cell, or introduced synthetic machinery, required toinitiate the specific transcription of a polynucleotide sequence.

The term “promoter/regulatory sequence” refers to a nucleic acidsequence which is required for expression of a gene product operablylinked to the promoter/regulatory sequence. In some instances, thissequence may be the core promoter sequence and in other instances, thissequence may also include an enhancer sequence and other regulatoryelements which are required for expression of the gene product. Thepromoter/regulatory sequence may, for example, be one which expressesthe gene product in a tissue specific manner.

The term “constitutive” promoter refers to a nucleotide sequence which,when operably linked with a polynucleotide which encodes or specifies agene product, causes the gene product to be produced in a cell undermost or all physiological conditions of the cell.

The term “inducible” promoter refers to a nucleotide sequence which,when operably linked with a polynucleotide which encodes or specifies agene product, causes the gene product to be produced in a cellsubstantially only when an inducer which corresponds to the promoter ispresent in the cell.

The term “tissue-specific” promoter refers to a nucleotide sequencewhich, when operably linked with a polynucleotide encodes or specifiedby a gene, causes the gene product to be produced in a cellsubstantially only if the cell is a cell of the tissue typecorresponding to the promoter.

The term “flexible polypeptide linker” as used in the context of a scFvrefers to a peptide linker that consists of amino acids such as glycineand/or serine residues used alone or in combination, to link variableheavy and variable light chain regions together. In one embodiment, theflexible polypeptide linker is a Gly/Ser linker and comprises the aminoacid sequence (Gly-Gly-Gly-Ser)_(n)(SEQ ID NO: 38), where n is apositive integer equal to or greater than 1. For example, n=1, n=2, n=3.n=4, n=5 and n=6, n=7, n=8, n=9 and n=10. In one embodiment, theflexible polypeptide linkers include, but are not limited to, (Gly₄Ser)₄ (SEQ ID NO: 27) or (Gly₄ Ser)₃ (SEQ ID NO: 28) In anotherembodiment, the linkers include multiple repeats of (Gly₂Ser), (GlySer)or (Gly₃Ser) (SEQ ID NO: 29). Also included within the scope of theinvention are linkers described in WO2012/138475, incorporated herein byreference).

As used herein, a 5′ cap (also termed an RNA cap, an RNA7-methylguanosine cap or an RNA m⁷G cap) is a modified guaninenucleotide that has been added to the “front” or 5′ end of a eukaryoticmessenger RNA shortly after the start of transcription. The 5′ capconsists of a terminal group which is linked to the first transcribednucleotide. Its presence is critical for recognition by the ribosome andprotection from RNases. Cap addition is coupled to transcription, andoccurs co-transcriptionally, such that each influences the other.Shortly after the start of transcription, the 5′ end of the mRNA beingsynthesized is bound by a cap-synthesizing complex associated with RNApolymerase. This enzymatic complex catalyzes the chemical reactions thatare required for mRNA capping. Synthesis proceeds as a multi-stepbiochemical reaction. The capping moiety can be modified to modulatefunctionality of mRNA such as its stability or efficiency oftranslation.

As used herein, “in vitro transcribed RNA” refers to RNA, preferablymRNA, that has been synthesized in vitro. Generally, the in vitrotranscribed RNA is generated from an in vitro transcription vector. Thein vitro transcription vector comprises a template that is used togenerate the in vitro transcribed RNA.

As used herein, a “poly(A)” is a series of adenosines attached bypolyadenylation to the mRNA. In the preferred embodiment of a constructfor transient expression, the polyA is between 50 and 5000 (SEQ ID NO:30), preferably greater than 64, more preferably greater than 100, mostpreferably greater than 300 or 400. poly(A) sequences can be modifiedchemically or enzymatically to modulate mRNA functionality such aslocalization, stability or efficiency of translation.

As used herein, “polyadenylation” refers to the covalent linkage of apolyadenylyl moiety, or its modified variant, to a messenger RNAmolecule. In eukaryotic organisms, most messenger RNA (mRNA) moleculesare polyadenylated at the 3′ end. The 3′ poly(A) tail is a long sequenceof adenine nucleotides (often several hundred) added to the pre-mRNAthrough the action of an enzyme, polyadenylate polymerase. In highereukaryotes, the poly(A) tail is added onto transcripts that contain aspecific sequence, the polyadenylation signal. The poly(A) tail and theprotein bound to it aid in protecting mRNA from degradation byexonucleases. Polyadenylation is also important for transcriptiontermination, export of the mRNA from the nucleus, and translation.Polyadenylation occurs in the nucleus immediately after transcription ofDNA into RNA, but additionally can also occur later in the cytoplasm.After transcription has been terminated, the mRNA chain is cleavedthrough the action of an endonuclease complex associated with RNApolymerase. The cleavage site is usually characterized by the presenceof the base sequence AAUAAA near the cleavage site. After the mRNA hasbeen cleaved, adenosine residues are added to the free 3′ end at thecleavage site.

As used herein, “transient” refers to expression of a non-integratedtransgene for a period of hours, days or weeks, wherein the period oftime of expression is less than the period of time for expression of thegene if integrated into the genome or contained within a stable plasmidreplicon in the host cell.

As used herein, the terms “treat”, “treatment” and “treating” refer tothe reduction or amelioration of the progression, severity and/orduration of a proliferative disorder, or the amelioration of one or moresymptoms (preferably, one or more discernible symptoms) of aproliferative disorder resulting from the administration of one or moretherapies (e.g., one or more therapeutic agents such as a CAR of theinvention). In specific embodiments, the terms “treat”, “treatment” and“treating” refer to the amelioration of at least one measurable physicalparameter of a proliferative disorder, such as growth of a tumor, notnecessarily discernible by the patient. In other embodiments the terms“treat”, “treatment” and “treating”-refer to the inhibition of theprogression of a proliferative disorder, either physically by, e.g.,stabilization of a discernible symptom, physiologically by, e.g.,stabilization of a physical parameter, or both. In other embodiments theterms “treat”, “treatment” and “treating” refer to the reduction orstabilization of tumor size or cancerous cell count.

The term “signal transduction pathway” refers to the biochemicalrelationship between a variety of signal transduction molecules thatplay a role in the transmission of a signal from one portion of a cellto another portion of a cell. The phrase “cell surface receptor”includes molecules and complexes of molecules capable of receiving asignal and transmitting signal across the membrane of a cell.

The term “subject” is intended to include living organisms in which animmune response can be elicited (e.g., mammals, human).

The term a “substantially purified” cell refers to a cell that isessentially free of other cell types. A substantially purified cell alsorefers to a cell which has been separated from other cell types withwhich it is normally associated in its naturally occurring state. Insome instances, a population of substantially purified cells refers to ahomogenous population of cells. In other instances, this term referssimply to cell that have been separated from the cells with which theyare naturally associated in their natural state. In some aspects, thecells are cultured in vitro. In other aspects, the cells are notcultured in vitro.

The term “therapeutic” as used herein means a treatment. A therapeuticeffect is obtained by reduction, suppression, remission, or eradicationof a disease state.

The term “prophylaxis” as used herein means the prevention of orprotective treatment for a disease or disease state.

The terms “cancer associated antigen” or “tumor antigen” interchangeablyrefers to a molecule (typically a protein, carbohydrate or lipid) thatis expressed on the surface of a cancer cell, either entirely or as afragment (e.g., MHC/peptide), and which is useful for the preferentialtargeting of a pharmacological agent to the cancer cell. In someembodiments, a tumor antigen is a marker expressed by both normal cellsand cancer cells, e.g., a lineage marker, e.g., CD19 on B cells. In someembodiments, a tumor antigen is a cell surface molecule that isoverexpressed in a cancer cell in comparison to a normal cell, forinstance, 1-fold over expression, 2-fold overexpression, 3-foldoverexpression or more in comparison to a normal cell. In someenbodiments, a tumor antigen is a cell surface molecule that isinappropriately synthesized in the cancer cell, for instance, a moleculethat contains deletions, additions or mutations in comparison to themolecule expressed on a normal cell. In some embodiments, a tumorantigen will be expressed exclusively on the cell surface of a cancercell, entirely or as a fragment (e.g., MHC/peptide), and not synthesizedor expressed on the surface of a normal cell. In some embodiments, theCARs of the present invention includes CARs comprising an antigenbinding domain (e.g., antibody or antibody fragment) that binds to a WICpresented peptide. Normally, peptides derived from endogenous proteinsfill the pockets of Major histocompatibility complex (WIC) class Imolecules, and are recognized by T cell receptors (TCRs) on CD8+Tlymphocytes. The MHC class I complexes are constitutively expressed byall nucleated cells. In cancer, virus-specific and/or tumor-specificpeptide/WIC complexes represent a unique class of cell surface targetsfor immunotherapy. TCR-like antibodies targeting peptides derived fromviral or tumor antigens in the context of human leukocyte antigen(HLA)-A1 or HLA-A2 have been described (see, e.g., Sastry et al., JVirol. 2011 85(5):1935-1942; Sergeeva et al., Blood, 2011117(16):4262-4272; Verma et al., J Immunol 2010 184(4):2156-2165;Willemsen et al., Gene Ther 2001 8(21):1601-1608; Dao et al., Sci TranslMed 2013 5(176):176ra33; Tassev et al., Cancer Gene Ther 201219(2):84-100). For example, TCR-like antibody can be identified fromscreening a library, such as a human scFv phage displayed library.

The term “transfected” or “transformed” or “transduced” refers to aprocess by which exogenous nucleic acid is transferred or introducedinto the host cell. A “transfected” or “transformed” or “transduced”cell is one which has been transfected, transformed or transduced withexogenous nucleic acid. The cell includes the primary subject cell andits progeny.

The term “specifically binds,” refers to an antibody, or a ligand, whichrecognizes and binds with a binding partner (e.g., tumor antigen)protein present in a sample, but which antibody or ligand does notsubstantially recognize or bind other molecules in the sample.

“Regulatable chimeric antigen receptor (RCAR),” as that term is usedherein, refers to a set of polypeptides, typically two in the simplestembodiments, which when in a RCARX cell, provides the RCARX cell withspecificity for a target cell, typically a cancer cell, and withregulatable intracellular signal generation or proliferation, which canoptimize an immune effector property of the RCARX cell. An RCARX cellrelies at least in part, on an antigen binding domain to providespecificity to a target cell that comprises the antigen bound by theantigen binding domain. In an embodiment, an RCAR includes adimerization switch that, upon the presence of a dimerization molecule,can couple an intracellular signaling domain to the antigen bindingdomain.

“Membrane anchor” or “membrane tethering domain”, as that term is usedherein, refers to a polypeptide or moiety, e.g., a myristoyl group,sufficient to anchor an extracellular or intracellular domain to theplasma membrane.

“Switch domain,” as that term is used herein, e.g., when referring to anRCAR, refers to an entity, typically a polypeptide-based entity, that,in the presence of a dimerization molecule, associates with anotherswitch domain. The association results in a functional coupling of afirst entity linked to, e.g., fused to, a first switch domain, and asecond entity linked to, e.g., fused to, a second switch domain. A firstand second switch domain are collectively referred to as a dimerizationswitch. In embodiments, the first and second switch domains are the sameas one another, e.g., they are polypeptides having the same primaryamino acid sequence, and are referred to collectively as ahomodimerization switch. In embodiments, the first and second switchdomains are different from one another, e.g., they are polypeptideshaving different primary amino acid sequences, and are referred tocollectively as a heterodimerization switch. In embodiments, the switchis intracellular. In embodiments, the switch is extracellular. Inembodiments, the switch domain is a polypeptide-based entity, e.g., FKBPor FRB-based, and the dimerization molecule is small molecule, e.g., arapalogue. In embodiments, the switch domain is a polypeptide-basedentity, e.g., an scFv that binds a myc peptide, and the dimerizationmolecule is a polypeptide, a fragment thereof, or a multimer of apolypeptide, e.g., a myc ligand or multimers of a myc ligand that bindto one or more myc scFvs. In embodiments, the switch domain is apolypeptide-based entity, e.g., myc receptor, and the dimerizationmolecule is an antibody or fragments thereof, e.g., myc antibody.

“Dimerization molecule,” as that term is used herein, e.g., whenreferring to an RCAR, refers to a molecule that promotes the associationof a first switch domain with a second switch domain. In embodiments,the dimerization molecule does not naturally occur in the subject, ordoes not occur in concentrations that would result in significantdimerization. In embodiments, the dimerization molecule is a smallmolecule, e.g., rapamycin or a rapalogue, e.g, RAD001.

The term “bioequivalent” refers to an amount of an agent other than thereference compound (e.g., RAD001), required to produce an effectequivalent to the effect produced by the reference dose or referenceamount of the reference compound (e.g., RAD001). In an embodiment theeffect is the level of mTOR inhibition, e.g., as measured by P70 S6kinase inhibition, e.g., as evaluated in an in vivo or in vitro assay,e.g., as measured by an assay described herein, e.g., the Boulay assay,or measurement of phosphorylated S6 levels by western blot. In anembodiment, the effect is alteration of the ratio of PD-1 positive/PD-1negative T cells, as measured by cell sorting. In an embodiment abioequivalent amount or dose of an mTOR inhibitor is the amount or dosethat achieves the same level of P70 S6 kinase inhibition as does thereference dose or reference amount of a reference compound. In anembodiment, a bioequivalent amount or dose of an mTOR inhibitor is theamount or dose that achieves the same level of alteration in the ratioof PD-1 positive/PD-1 negative T cells as does the reference dose orreference amount of a reference compound.

The term ‘low, immune enhancing, dose” when used in conjuction with anmTOR inhibitor, e.g., an allosteric mTOR inhibitor, e.g., RAD001 orrapamycin, or a catalytic mTOR inhibitor, refers to a dose of mTORinhibitor that partially, but not fully, inhibits mTOR activity, e.g.,as measured by the inhibition of P70 S6 kinase activity. Methods forevaluating mTOR activity, e.g., by inhibition of P70 S6 kinase, arediscussed herein. The dose is insufficient to result in complete immunesuppression but is sufficient to enhance the immune response. In anembodiment, the low, immune enhancing, dose of mTOR inhibitor results ina decrease in the number of PD-1 positive T cells and/or an increase inthe number of PD-1 negative T cells, or an increase in the ratio of PD-1negative T cells/PD-1 positive T cells. In an embodiment, the low,immune enhancing, dose of mTOR inhibitor results in an increase in thenumber of naive T cells. In an embodiment, the low, immune enhancing,dose of mTOR inhibitor results in one or more of the following:

an increase in the expression of one or more of the following markers:CD62L^(high), CD127^(high), CD27⁺, and BCL2, e.g., on memory T cells,e.g., memory T cell precursors;

a decrease in the expression of KLRG1, e.g., on memory T cells, e.g.,memory T cell precursors; and

an increase in the number of memory T cell precursors, e.g., cells withany one or combination of the following characteristics: increasedCD62L^(high), increased CD127^(high), increased CD27⁺, decreased KLRG1,and increased BCL2;

wherein any of the changes described above occurs, e.g., at leasttransiently, e.g., as compared to a non-treated subject.

Ranges: throughout this disclosure, various aspects of the invention canbe presented in a range format. It should be understood that thedescription in range format is merely for convenience and brevity andshould not be construed as an inflexible limitation on the scope of theinvention. Accordingly, the description of a range should be consideredto have specifically disclosed all the possible subranges as well asindividual numerical values within that range. For example, descriptionof a range such as from 1 to 6 should be considered to have specificallydisclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numberswithin that range, for example, 1, 2, 2.7, 3, 4, 5, 5.3, and 6. Asanother example, a range such as 95-99% identity, includes somethingwith 95%, 96%, 97%, 98%, or 99% identity, and includes subranges such as96-99%, 96-98%, 96-97%, 97-99%, 97-98%, and 98-99% identity. Thisapplies regardless of the breadth of the range.

DESCRIPTION

Provided herein are compositions of matter and methods of use for thetreatment of a disease such as cancer using anti-mesothelin chimericantigen receptors (CAR), e.g., human mesothelin CAR.

In one aspect, the invention provides a number of chimeric antigenreceptors comprising an antibody or antibody fragment engineered forspecific binding to a mesothelin protein. In one aspect, the inventionprovides a cell (e.g., T cell or NK cell) engineered to express a CAR,e.g., wherein the CAR T cell (“CART”) exhibits an anticancer property.In one aspect a cell is transformed with the CAR and the CAR isexpressed on the cell surface. In some embodiments, the cell (e.g., Tcell or NK cell) is transduced with a viral vector encoding a CAR. Insome embodiments, the viral vector is a retroviral vector. In someembodiments, the viral vector is a lentiviral vector. In some suchembodiments, the cell may stably express the CAR. In another embodiment,the cell (e.g., T cell or NK cell) is transfected with a nucleic acid,e.g., mRNA, cDNA, DNA, encoding a CAR. In some such embodiments, thecell may transiently express the CAR.

In one aspect, the mesothelin protein binding portion of the CAR is ascFv antibody fragment. In one aspect such antibody fragments arefunctional in that they retain the equivalent binding affinity, i.e.,they bind the same antigen with comparable affinity, as the IgG antibodyfrom which it is derived. In one aspect such antibody fragments arefunctional in that they provide a biological response that can include,but is not limited to, activation of an immune response, inhibition ofsignal-transduction origination from its target antigen, inhibition ofkinase activity, and the like, as will be understood by a skilledartisan. In one aspect, the mesothelin antigen binding domain of the CARis a scFv antibody fragment that is human or humanized compared to themurine sequence of the scFv from which it is derived. In one embodiment,the human anti-mesothelin scFv antibody fragment comprises a light chainvariable region and/or a heavy chain variable region provided in Table2, or a sequence with substantial identity thereto, e.g., 95-99%identity.

In some aspects, the antibodies of the invention are incorporated into achimeric antigen receptor (CAR). In one aspect, the CAR comprises thepolypeptide sequence provided herein as SEQ ID NO: 39; SEQ ID NO: 40,SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO:45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ IDNO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59,SEQ ID NO: 60, SEQ ID NO: 61, and SEQ ID NO: 62, or a sequence with95-99% identify thereof.

In one aspect, the human scFv portion of a CAR is encoded by a transgenewhose sequence has been codon optimized for expression in a mammaliancell. In one aspect, entire CAR construct of the invention is encoded bya transgene whose entire sequence has been codon optimized forexpression in a mammalian cell. Codon optimization refers to thediscovery that the frequency of occurrence of synonymous codons (i.e.,codons that code for the same amino acid) in coding DNA is biased indifferent species. Such codon degeneracy allows an identical polypeptideto be encoded by a variety of nucleotide sequences. A variety of codonoptimization methods is known in the art, and include, e.g., methodsdisclosed in at least U.S. Pat. Nos. 5,786,464 and 6,114,148.

In one aspect, the human mesothelin CAR molecule comprises the scFvportion provided in SEQ ID NO: 39. In one aspect, the human mesothelinCAR molecule comprises the scFv portion provided in SEQ ID NO: 40. Inone aspect, the human mesothelin CAR molecule comprises the scFv portionprovided in SEQ ID NO: 41. In one aspect, the human mesothelin CARmolecule comprises the scFv portion provided in SEQ ID NO: 42. In oneaspect, the human mesothelin CAR molecule comprises the scFv portionprovided in SEQ ID NO: 43. In one aspect, the human mesothelin CARmolecule comprises the scFv portion provided in SEQ ID NO: 44. In oneaspect, the human mesothelin CAR molecule comprises the scFv portionprovided in SEQ ID NO: 45. In one aspect, the human mesothelin CARmolecule comprises the scFv portion provided in SEQ ID NO: 46. In oneaspect, the human mesothelin CAR molecule comprises the scFv portionprovided in SEQ ID NO: 47. In one aspect, the human mesothelin CARmolecule comprises the scFv portion provided in SEQ ID NO: 48. In oneaspect, the human mesothelin CAR molecule comprises the scFv portionprovided in SEQ ID NO: 49. In one aspect, the human mesothelin CARmolecule comprises the scFv portion provided in SEQ ID NO: 50. In oneaspect, the human mesothelin CAR molecule comprises the scFv portionprovided in SEQ ID NO: 51. In one aspect, the human mesothelin CARmolecule comprises the scFv portion provided in SEQ ID NO: 52. In oneaspect, the human mesothelin CAR molecule comprises the scFv portionprovided in SEQ ID NO: 53. In one aspect, the human mesothelin CARmolecule comprises the scFv portion provided in SEQ ID NO: 54. In oneaspect, the human mesothelin CAR molecule comprises the scFv portionprovided in SEQ ID NO: 55. In one aspect, the human mesothelin CARmolecule comprises the scFv portion provided in SEQ ID NO: 56. In oneaspect, the human mesothelin CAR molecule comprises the scFv portionprovided in SEQ ID NO: 57. In one aspect, the human mesothelin CARmolecule comprises the scFv portion provided in SEQ ID NO: 58. In oneaspect, the human mesothelin CAR molecule comprises the scFv portionprovided in SEQ ID NO: 59. In one aspect, the human mesothelin CARmolecule comprises the scFv portion provided in SEQ ID NO: 60. In oneaspect, the human mesothelin CAR molecule comprises the scFv portionprovided in SEQ ID NO: 61. In one aspect, the human mesothelin CARmolecule comprises the scFv portion provided in SEQ ID NO: 62.

In one aspect, the CAR disclosed herein combine an antigen bindingdomain of a specific antibody with an intracellular signaling molecule.For example, in some aspects, the intracellular signaling moleculeincludes, but is not limited to, CD3-zeta chain, 4-1BB and CD28signaling modules and combinations thereof. In one aspect, the antigenbinding domain binds to mesothelin. In one aspect, the -mesothelin CARcomprises the sequence provided in Table 2.

In one aspect, the mesothelin CAR comprises a CAR selected from thesequence provided in one or more of SEQ ID NOS: 63-86. In one aspect,themesothelin CAR comprises the sequence provided in SEQ ID NO: 63. Inone aspect, themesothelin CAR comprises the sequence provided in SEQ IDNO: 64. In one aspect, themesothelin CAR comprises the sequence providedin SEQ ID NO: 65. In one aspect, themesothelin CAR comprises thesequence provided in SEQ ID NO: 66. In one aspect, themesothelin CARcomprises the sequence provided in SEQ ID NO: 67. In one aspect,themesothelin CAR comprises the sequence provided in SEQ ID NO: 68. Inone aspect, themesothelin CAR comprises the sequence provided in SEQ IDNO: 69. In one aspect, themesothelin CAR comprises the sequence providedin SEQ ID NO: 70. In one aspect, themesothelin CAR comprises thesequence provided in SEQ ID NO: 71. In one aspect, themesothelin CARcomprises the sequence provided in SEQ ID NO: 72. In one aspect,themesothelin CAR comprises the sequence provided in SEQ ID NO: 73. Inone aspect, themesothelin CAR comprises the sequence provided in SEQ IDNO: 74. In one aspect, themesothelin CAR comprises the sequence providedin SEQ ID NO: 75. In one aspect, themesothelin CAR comprises thesequence provided in SEQ ID NO: 76. In one aspect, themesothelin CARcomprises the sequence provided in SEQ ID NO: 77. In one aspect,themesothelin CAR comprises the sequence provided in SEQ ID NO: 78. Inone aspect, themesothelin CAR comprises the sequence provided in SEQ IDNO: 79. In one aspect, themesothelin CAR comprises the sequence providedin SEQ ID NO: 80. In one aspect, themesothelin CAR comprises thesequence provided in SEQ ID NO: 81. In one aspect, themesothelin CARcomprises the sequence provided in SEQ ID NO: 82. In one aspect,themesothelin CAR comprises the sequence provided in SEQ ID NO: 83. Inone aspect, themesothelin CAR comprises the sequence provided in SEQ IDNO: 84. In one aspect, themesothelin CAR comprises the sequence providedin SEQ ID NO: 85. In one aspect, themesothelin CAR comprises thesequence provided in SEQ ID NO: 86.

Furthermore, the present invention provides mesothelin CAR compositionsand their use in medicaments or methods for treating, among otherdiseases, cancer or any malignancy or autoimmune diseases involvingcells or tissues which express mesothelin.

In one aspect, the invention provides a cell (e.g., T cell or NK cell)engineered to express a chimeric antigen receptor (CAR), wherein the CART cell (“CART”) exhibits an antitumor property. A preferred antigen ismesothelin. In one aspect, the antigen binding domain of the CARcomprises a human anti-mesothelin antibody fragment. In one aspect, theantigen binding domain of the CAR comprises a human anti-mesothelinantibody fragment comprising an scFv. Accordingly, the inventionprovides an mesothelin CAR that comprises a human anti-mesothelinbinding domain and is engineered into a T cell or NK cell and methods oftheir use for adoptive therapy.

In one aspect, mesothelin CAR comprises at least one intracellularsignaling domain selected from the group consisting of a CD137 (4-1BB)signaling domain, a CD28 signaling domain, a CD3zeta signal domain, andany combination thereof. In one aspect, the mesothelin CAR comprises atleast one intracellular signaling domain of one or more costimulatorymolecule(s) other than a CD137 (4-1BB) or CD28, a CD3zeta signal domain,and any combination thereof.

Furthermore, the present invention provides mesothelin CAR compositionsand their use in medicaments or methods for treating, among otherdiseases, cancer or any malignancy or autoimmune diseases involvingcells or tissues which express mesothelin.

Chimeric Antigen Receptor (CAR)

The present invention encompasses a recombinant nucleic acid constructcomprising sequences encoding a CAR, wherein the CAR comprises anantibody that binds specifically to mesothelin, e.g., a human antibodyfragment that specifically binds to mesothelin. In one aspect, themesothelin is human mesothelin, and the sequence of the antibodyfragment is contiguous with, and in the same reading frame as a nucleicacid sequence encoding an intracellular signaling domain. Theintracellular signaling domain can comprise a costimulatory signalingdomain and/or a primary signaling domain, e.g., a zeta chain. Thecostimulatory signaling domain refers to a portion of the CAR comprisingat least a portion of the intracellular domain of a costimulatorymolecule.

In specific aspects, a CAR construct of the invention comprises a scFvdomain selected from the group consisting of SEQ ID NOS: 39-62, whereinthe scFv may be preceded by an optional leader sequence such as providedin SEQ ID NO: 1, and followed by an optional hinge sequence such asprovided in SEQ ID NO:2 or SEQ ID NO: 3 or SEQ ID NO:4 or SEQ ID NO:5, atransmembrane region such as provided in SEQ ID NO:6, an intracellularsignalling domain that includes SEQ ID NO:7 or SEQ ID NO:8 and a CD3zeta sequence that includes SEQ ID NO:9 or SEQ ID NO: 10, wherein thedomains are contiguous with and in the same reading frame to form asingle fusion protein. Also included in the invention is a nucleotidesequence that encodes the polypeptide selected from the group consistingof SEQ ID NO: 87; SEQ ID NO: 88, SEQ ID NO: 89, SEQ ID NO: 90, SEQ IDNO: 91, SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 95, SEQID NO: 96, SEQ ID NO: 97, SEQ ID NO: 98, SEQ ID NO: 99, SEQ ID NO: 100,SEQ ID NO: 101, SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104, SEQ IDNO: 105, SEQ ID NO: 106, SEQ ID NO: 107, SEQ ID NO: 108, SEQ ID NO: 109,and SEQ ID NO: 110, or a sequence with 95-99% identify thereof. Alsoincluded in the invention is a nucleotide sequence that encodes thepolypeptide of each of the scFv fragments selected from the groupconsisting of SEQ ID NO: 39; SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO:42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ IDNO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56,SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO:61, and SEQ ID NO: 62, or a sequence with 95-99% identify thereof, andeach of the domains of SEQ ID NOS: 1, 2, and 6-9, plus the encodedmesothelin CAR fusion protein of the invention. In one aspect anexemplary mesothelin CAR constructs comprise an optional leadersequence, an extracellular mesothelin binding domain, a hinge, atransmembrane domain, and an intracellular stimulatory domain. In oneaspect, the mesothelin CAR construct comprises an optional leadersequence, a mesothelin binding domain, a hinge, a transmembrane domain,an intracellular costimulatory domain and an intracellular stimulatorydomain. Specific mesothelin CAR constructs containing human scFv domainsare provided as SEQ ID NOs: 87-110.

Full-length CAR sequences are also provided herein as SEQ ID NO: 63; SEQID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68,SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO: 71, SEQ ID NO: 72, SEQ ID NO:73, SEQ ID NO: 74, SEQ ID NO: 75, SEQ ID NO: 76, SEQ ID NO: 77, SEQ IDNO: 78, SEQ ID NO: 79, SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 82, SEQID NO: 83, SEQ ID NO: 84, SEQ ID NO: 85, or SEQ ID NO: 86. An exemplaryleader sequence is provided as SEQ ID NO: 1. An exemplary hinge/spacersequence is provided as SEQ ID NO: 2 or SEQ ID NO:3 or SEQ ID NO:4 orSEQ ID NO:5. An exemplary transmembrane domain sequence is provided asSEQ ID NO:6. An exemplary sequence of the intracellular signaling domainof the 4-1BB protein is provided as SEQ ID NO: 7. An exemplary sequenceof the intracellular signaling domain of CD27 is provided as SEQ IDNO:8. An exemplary CD3zeta domain sequence is provided as SEQ ID NO: 9or SEQ ID NO:10.

In one aspect, the present invention encompasses a recombinant nucleicacid construct comprising a nucleic acid molecule encoding a CAR,wherein the nucleic acid molecule comprises the nucleic acid sequenceencoding an anti-mesothelin binding domain, e.g., described herein, thatis contiguous with and in the same reading frame as a nucleic acidsequence encoding an intracellular signaling domain. In one aspect, theanti-mesothlin binding domain is selected from one or more of SEQ IDNOS: 87-110. In one aspect, the anti-mesothelin binding domain comprisesSEQ ID NO: 87. In one aspect, the anti-mesothelin binding domaincomprises SEQ ID NO: 88. In one aspect, the anti-mesothelin bindingdomain comprises SEQ ID NO: 89. In one aspect, the anti-mesothelinbinding domain comprises SEQ ID NO: 90. In one aspect, theanti-mesothelin binding domain comprises SEQ ID NO: 91. In one aspect,the anti-mesothelin binding domain comprises SEQ ID NO: 92. In oneaspect, the anti-mesothelin binding domain comprises SEQ ID NO: 93. Inone aspect, the anti-mesothelin binding domain comprises SEQ ID NO: 94.In one aspect, the anti-mesothelin binding domain comprises SEQ ID NO:95. In one aspect, the anti-mesothelin binding domain comprises SEQ IDNO: 96. In one aspect, the anti-mesothelin binding domain comprises SEQID NO: 97. In one aspect, the anti-mesothelin binding domain comprisesSEQ ID NO: 98. In one aspect, the anti-mesothelin binding domaincomprises SEQ ID NO: 99. In one aspect, the anti-mesothelin bindingdomain comprises SEQ ID NO: 100. In one aspect, the anti-mesothelinbinding domain comprises SEQ ID NO: 101. In one aspect, theanti-mesothelin binding domain comprises SEQ ID NO: 102. In one aspect,the anti-mesothelin binding domain comprises SEQ ID NO: 103. In oneaspect, the anti-mesothelin binding domain comprises SEQ ID NO: 104. Inone aspect, the anti-mesothelin binding domain comprises SEQ ID NO: 105.In one aspect, the anti-mesothelin binding domain comprises SEQ ID NO:106. In one aspect, the anti-mesothelin binding domain comprises SEQ IDNO: 107. In one aspect, the anti-mesothelin binding domain comprises SEQID NO: 108. In one aspect, the anti-mesothelin binding domain comprisesSEQ ID NO: 109. In one aspect, the anti-mesothelin binding domaincomprises SEQ ID NO: 110. In one aspect, the present inventionencompasses a recombinant DNA construct comprising a transgene encodinga CAR, wherein the transgene comprises the nucleic acid sequenceencoding an anti-mesothelin binding domain described herein, e.g., ahuman anti-mesothelin binding domain selected from one or more of SEQ IDNOS:87-110, wherein the sequence is contiguous with and in the samereading frame as the nucleic acid sequence encoding an intracellularsignaling domain. An exemplary intracellular signaling domain that canbe used in the CAR includes, but is not limited to, one or moreintracellular signaling domains of, e.g., CD3-zeta, CD28, 4-1BB, and thelike. In some instances, the CAR can comprise any combination ofCD3-zeta, CD28, 4-1BB, and the like. In one aspect the nucleic acidsequence of a CAR construct of the invention is selected from one ormore of SEQ ID NOS: 111-134. In one aspect the nucleic acid sequence ofa CAR construct is SEQ ID NO: 111. In one aspect the nucleic acidsequence of a CAR construct is SEQ ID NO: 112. In one aspect the nucleicacid sequence of a CAR construct is SEQ ID NO: 113. In one aspect thenucleic acid sequence of a CAR construct is SEQ ID NO: 114. In oneaspect the nucleic acid sequence of a CAR construct is SEQ ID NO: 115.In one aspect the nucleic acid sequence of a CAR construct is SEQ ID NO:116. In one aspect the nucleic acid sequence of a CAR construct is SEQID NO: 117. In one aspect the nucleic acid sequence of a CAR constructis SEQ ID NO: 118. In one aspect the nucleic acid sequence of a CARconstruct is SEQ ID NO: 119. In one aspect the nucleic acid sequence ofa CAR construct is SEQ ID NO: 120. In one aspect the nucleic acidsequence of a CAR construct is SEQ ID NO: 121. In one aspect the nucleicacid sequence of a CAR construct is SEQ ID NO: 122. In one aspect thenucleic acid sequence of a CAR construct is SEQ ID NO: 123. In oneaspect the nucleic acid sequence of a CAR construct is SEQ ID NO: 124.In one aspect the nucleic acid sequence of a CAR construct is SEQ ID NO:125. In one aspect the nucleic acid sequence of a CAR construct is SEQID NO: 126. In one aspect the nucleic acid sequence of a CAR constructis SEQ ID NO: 127. In one aspect the nucleic acid sequence of a CARconstruct is SEQ ID NO: 128. In one aspect the nucleic acid sequence ofa CAR construct is SEQ ID NO: 129. In one aspect the nucleic acidsequence of a CAR construct is SEQ ID NO: 130. In one aspect the nucleicacid sequence of a CAR construct is SEQ ID NO: 131. In one aspect thenucleic acid sequence of a CAR construct is SEQ ID NO: 132. In oneaspect the nucleic acid sequence of a CAR construct is SEQ ID NO: 133.In one aspect the nucleic acid sequence of a CAR construct is SEQ ID NO:134.

The nucleic acid sequences coding for the desired molecules can beobtained using recombinant methods known in the art, such as, forexample by screening libraries from cells expressing the gene, byderiving the gene from a vector known to include the same, or byisolating directly from cells and tissues containing the same, usingstandard techniques. Alternatively, the nucleic acid of interest can beproduced synthetically, rather than cloned.

The present invention includes retroviral and lentiviral vectorconstructs expressing a CAR that can be directly transduced into a cell.The present invention also includes an RNA construct that can bedirectly transfected into a cell. A method for generating mRNA for usein transfection involves in vitro transcription (IVT) of a template withspecially designed primers, followed by polyA addition, to produce aconstruct containing 3′ and 5′ untranslated sequence (“UTR”), a 5′ capand/or Internal Ribosome Entry Site (IRES), the nucleic acid to beexpressed, and a polyA tail, typically 50-2000 bases in length (SEQ IDNO: 35). RNA so produced can efficiently transfect different kinds ofcells. In one embodiment, the template includes sequences for the CAR.In an embodiment, an RNA CAR vector is transduced into a T cell byelectroporation.

Antigen Binding Domain

In one aspect, the CAR of the invention comprises a target-specificbinding element otherwise referred to as an antigen binding domain. Thechoice of antigen binding domain depends upon the type and number ofantigens that define the surface of a target cell. For example, theantigen binding domain may be chosen to recognize a antigen that acts asa cell surface marker on target cells associated with a particulardisease state.

In one aspect, the CAR-mediated immune effector cell response can bedirected to cells that express an antigen of interest, where the CARcomprises an antigen binding domain that specifically binds to theantigen of interest. In one aspect, the portion of the CAR comprisingthe antigen binding domain comprises an antigen binding domain thattargets mesothelin. In one aspect, the antigen binding domain targetshuman mesothelin.

The antigen binding domain can be any domain that binds to the antigenincluding but not limited to a monoclonal antibody, a polyclonalantibody, a recombinant antibody, a human antibody, a humanizedantibody, and a functional fragment thereof, including but not limitedto a single-domain antibody such as a heavy chain variable domain (VH),a light chain variable domain (VL) and a variable domain (VHH) ofcamelid derived nanobody, and to an alternative scaffold known in theart to function as an antigen binding domain, such as a recombinantfibronectin domain, and the like. In some instances, it is beneficialfor the antigen binding domain to be derived from the same species inwhich the CAR will ultimately be used in. For example, for use inhumans, it may be beneficial for the antigen binding domain of the CARto comprise human or humanized residues for the antigen binding domainof an antibody or antibody fragment. Thus, in one aspect, the antigenbinding domain comprises a human antibody or an antibody fragment.

In one embodiment, the anti-mesothelin binding domain does not compete,or competes poorly, for binding to human mesothelin with an antigenbinding domain comprising an amino acid sequence comprising SEQ ID NO:279, e.g., murine SS1 scFv, e.g., in a competition assay describedherein.

The amino acid sequence of murine SS1 scFv is provided below (SEQ ID NO:279):

QVQLQQSGPELEKPGASVKISCKASGYSFTGYTMNWVKQSHGKSLEWIGLITPYNGASSYNQKFRGKATLTVDKSSSTAYMDLLSLTSEDSAVYFCARGGYDGRGFDYWGQGTTVTVSSGGGGSGGGGSGGGGSDIELTQSPAIMSASPGEKVTMTCSASSSVSYMHWYQQKSGTSPKRWIYDTSKLASGVPGRFSGSGSGNSYSLTISSVEAEDDATYYCQQWSGYPLTFGAGTKLEI

In one embodiment, the anti-mesothelin binding domain competes forbinding to human mesothelin with an antigen binding domain comprising aLC CDR1, LC CDR2 and LC CDR3 of an anti-mesothelin light chain aminoacid sequence selected from SEQ ID NO: 43 or SEQ ID NO: 49 and an HCCDR1, HC CDR2, and HC CDR3 of an anti-mesothelin heavy chain amino acidsequence selected from SEQ ID NO: 43 or SEQ ID NO: 49, e.g., in acompetition assay described herein. In one embodiment, theanti-mesothelin binding domain competes for binding to human mesothelinwith an antigen binding domain comprising a LC CDR1 selected from SEQ IDNO: 203 or SEQ ID NO: 209, a LC CDR2 selected from SEQ ID NO: 227 or SEQID NO: 233, and a LC CDR3 selected from SEQ ID NO: 251 or SEQ ID NO:257; and a HC CDR1 selected from SEQ ID NO: 138 or SEQ ID NO: 144, a HCCDR2 selected from SEQ ID NO: 156 or SEQ ID NO: 162, and a HC CDR3selected from SEQ ID NO: 179 or SEQ ID NO: 185, e.g., in a competitionassay described herein.

In one embodiment, the anti-mesothelin binding domain competes forbinding to human mesothelin with an antigen binding domain comprising asequence selected from SEQ ID NO: 43 or SEQ ID NO: 49, e.g., in acompetition assay described herein.

In embodiments, the competition assay is an SPR-based assay. Briefly,the antigen, e.g., human mesothelin, is immobilized on a surface.Through a microflow system, a reference antibody is injected over theantigen layer. Upon binding of the reference antibody to the antigen, anincrease in signal, typically expressed in response units (RU) isdetected, e.g., reference signal. After a desired time, a test antibodyis injected over the antigen layer. If the test antibody binds to adifferent region or epitope of the antigen, then an additional increasein signal is detected, e.g., a 5% or more, 10% or more, 15% or more, 20%or more, 25% or more, 30% or more, 35%, or more, 40% or more, 45% ormore, 50% or more, 55% of more, 60% or more, 65% or more, 70% or more,75% or more, 80% or more, 85% or more, 90% or more, or 95% or moreincrease in signal, e.g., RU, as compared to the highest signal detectedupon binding of the reference antibody, e.g., the reference signal. Ifthe test antibody binds to the same region or epitope of the antigen,then little or no increase in signal, e.g., RU, will be detected, e.g.,less than 20%, less than 15%, less than 10%, less than 5%, less than 4%,less than 3%, less than 2%, or less than 1% increase in signal, e.g.,RU, as compared to the highest signal detected upon binding of thereference antibody, e.g., the reference signal. When using thisSPR-based competition assay, an antibody is said to compete with thereference antibody when less than 20%, less than 15%, less than 10%,less than 5%, less than 4%, less than 3%, less than 2%, or less than 1%increase in signal, e.g., RU, is detected when compared to the referencesignal detected upon binding of the reference antibody to the antigen.An antibody is said to not compete, or compete poorly, with a referenceantibody when a 5% or more, 10% or more, 15% or more, 20% or more, 25%or more, 30% or more, 35%, or more, 40% or more, 45% or more, 50% ormore, 55% of more, 60% or more, 65% or more, 70% or more, 75% or more,80% or more, 85% or more, 90% or more, or 95% or more increase insignal, e.g., RU, is detected when compared to the reference signaldetected upon binding of the reference antibody to the antigen.

Identification of the epitope bound by the antigen binding domainsdescribed herein can be determined by various methods known in the art.For example, crystal structures can be generated containing the antigenbinding domain bound to, or in complex with, the antigen. In anotherexample, assays, e.g., a protection assay, can be performed to identifythe regions of the antigen contribute to the epitope, or to identify theepitope. An exemplary protection assay, a hydrogen/deuterium exchange(HDX) mass spectrometry assay, is described further in Example 18. TheHDX mass spectrometry was performed to identify the putative epitopes onhuman MSLN, e.g., hMSLN₂₉₆₋₅₈₈, e.g., SEQ ID NO: 278, for murine SS1,e.g., SEQ ID NO: 279, and the M5 scFv described herein, e.g., SEQ ID NO:43. hMSLN₂₉₆₋₅₈₈, e.g., SEQ ID NO: 278, represents amino acids 296-588of human mesothelin, e.g., the first amino acid of SEQ ID NO: 278 isamino acid 296 and the last amino acid of SEQ ID NO: 278 is amino acid588. The amino acid sequence for human mesothelin, amino acids 296-588is provided below: (SEQ ID NO: 278)

EVEKTACPSGKKAREIDESLIFYKKWELEACVDAALLATQMDRVNAIPFTYEQLDVLKHKLDELYPQGYPESVIQHLGYLFLKMSPEDIRKWNVTSLETLKALLEVNKGHEMSPQAPRRPLPQVATLIDRFVKGRGQLDKDTLDTLTAFYPGYLCSLSPEELSSVPPSSIWAVRPQDLDTCDPRQLDVLYPKARLAFQNMNGSEYFVKIQSFLGGAPTEDLKALSQQNVSMDLATFMKLRTDAVLPLTVAEVQKLLGPHVEGLKAEERHRPVRDWILRQRQDDLDTLGLGLQG

The results of the HDX mass spectrometry assay indicated that one ormore amino acids of 314-315, 317-318, 346-349, and 369-375 ofhMSLN₂₉₆₋₅₈₈, e.g., SEQ ID NO: 278 contribute to the epitope recognizedby SS1. The results of the HDX mass spectrometry assay indicated thatone or more amino acids of 485-490, 498-507, 532-537, or 545-572 ofhMSLN₂₉₆₋₅₈₈, e.g., SEQ ID NO: 278, contribute to the epitope recognizedby an anti-mesothelin antigen binding domain described herein, e.g., M5scFv, e.g., SEQ ID NO: 43.

In one embodiment, the anti-mesothelin binding domain described hereinbinds to a different epitope of human mesothelin, e.g., SEQ ID NO: 278,than the epitope of human mesothelin targeted by the antigen bindingdomain comprising a sequence comprising SEQ ID NO: 279, e.g., murineSS1.

In one embodiment, the epitope recognized by SS1 comprises a sequenceselected from amino acids 314-315, 317-318, 346-349, or 369-375 ofhMSLN₂₉₆₋₅₈₈, e.g., SEQ ID NO: 278, or any combination thereof. In oneembodiment, the epitope recognized by SS1 comprises one or more aminoacids selected from amino acids 314-315, 317-318, 346-349, or 369-375 ofhMSLN₂₉₆₋₅₈₈, e.g., SEQ ID NO: 278.

In one embodiment, the anti-mesothelin binding domain described hereinbinds to the C-terminus of human mesothelin. In one embodiment, theanti-mesothelin binding domain described herein binds an epitope withinamino acids 450-588 of SEQ ID NO: 278, e.g., wherein the epitope, inpart or in whole, can be found within amino acids 450-588, within aminoacids 480-580, or within amino acids 485-572 of SEQ ID NO: 278. In oneembodiment, the epitope recognized by an anti-mesothelin binding domaindescribed herein comprises a sequence selected from amino acids 485-490,498-507, 532-537, or 545-572 of hMSLN₂₉₆₋₅₈₈, e.g., SEQ ID NO: 278, orany combination thereof. In one embodiment, the epitope recognized by ananti-mesothelin binding domain described herein comprises one or moreamino acids selected from 485-490, 498-507, 532-537, or 545-572 ofhMSLN₂₉₆₋₅₈₈, e.g., SEQ ID NO: 278, or any combination thereof.

In one embodiment, the anti-mesothelin binding domain comprises one ormore (e.g., all three) light chain complementary determining region 1(LC CDR1), light chain complementary determining region 2 (LC CDR2), andlight chain complementary determining region 3 (LC CDR3) of a humananti-mesothelin binding domain selected from SEQ ID NOS: 39-62 and oneor more (e.g., all three) heavy chain complementary determining region 1(HC CDR1), heavy chain complementary determining region 2 (HC CDR2), andheavy chain complementary determining region 3 (HC CDR3) of a humananti-mesothelin binding domain selected from SEQ ID NOS: 39-62 In oneembodiment, the human anti-mesothelin binding domain comprises a lightchain variable region described herein (e.g., in Table 2) and/or a heavychain variable region described herein (e.g., in Table 2). In oneembodiment, the anti-mesothelin binding domain is a scFv comprising alight chain variable region and a heavy chain variable region of anamino acid sequence of Table 2. In an embodiment, the anti-mesothelinbinding domain (e.g., an scFV) comprises: a light chain variable regioncomprising an amino acid sequence having at least one, two or threemodifications (e.g., substitutions) but not more than 30, 20 or 10modifications (e.g., substitutions) of an amino acid sequence of a lightchain variable region provided in Table 2, or a sequence with 95-99%identity to an amino acid sequence of Table 2; and/or a heavy chainvariable region comprising an amino acid sequence having at least one,two or three modifications (e.g., substitutions) but not more than 30,20 or 10 modifications (e.g., substitutions) of an amino acid sequenceof a heavy chain variable region provided in Table 2, or a sequence with95-99% identity to an amino acid sequence of Table 2.

In one embodiment, the human anti-mesothelin binding domain comprises asequence selected from a group consisting of SEQ ID NOS: 39-62, or asequence with 95-99% identify thereof. In one embodiment, the nucleicacid sequence encoding the human anti-mesothelin binding domaincomprises a sequence selected from a group consisting of SEQ ID NO:87-110, or a sequence with 95-99% identify thereof. In one embodiment,the human anti-mesothelin binding domain is a scFv, and a light chainvariable region comprising an amino acid sequence described herein,e.g., in Table 2 or 3, is attached to a heavy chain variable regioncomprising an amino acid sequence described herein, e.g., in Table 2 or3, via a linker, e.g., a linker described herein. In one embodiment, thehumanized anti-mesothelin binding domain includes a (Gly4-Ser)n linker(SEQ ID NO: 26), wherein n is 1, 2, 3, 4, 5, or 6, preferably 3 or 4.The light chain variable region and heavy chain variable region of ascFv can be, e.g., in any of the following orientations: light chainvariable region-linker-heavy chain variable region or heavy chainvariable region-linker-light chain variable region.

In one aspect, the antigen binding domain portion comprises one or moresequence selected from SEQ ID NOS:39-62. In one aspect the CAR isselected from one or more sequence selected from SEQ ID NOS: 63-86.

In one aspect, the antibodies of the invention may exist in a variety ofother forms including, for example, Fab, Fab′, F(ab′)₂, Fv fragments,scFv antibody fragments, disulfide-linked Fvs (sdFv), a Fd fragmentconsisting of the VH and CH1 domains, linear antibodies, single domainantibodies such as sdAb (either VL or VH), camelid VHH domains,multi-specific antibodies formed from antibody fragments such as abivalent fragment comprising two Fab fragments linked by a disulfidebrudge at the hinge region, and an isolated CDR or other epitope bindingfragments of an antibody. In one aspect, the antibody fragment providedherein is a scFv. In some instances, a human scFv may also be derivedfrom a yeast display library.

A display library is a collection of entities; each entity includes anaccessible polypeptide component and a recoverable component thatencodes or identifies the polypeptide component. The polypeptidecomponent is varied so that different amino acid sequences arerepresented. The polypeptide component can be of any length, e.g. fromthree amino acids to over 300 amino acids. A display library entity caninclude more than one polypeptide component, for example, the twopolypeptide chains of a Fab. In one exemplary embodiment, a displaylibrary can be used to identify an anti-mesothelin binding domain. In aselection, the polypeptide component of each member of the library isprobed with mesothelin, or a fragment there, and if the polypeptidecomponent binds to the mesothelin, the display library member isidentified, typically by retention on a support.

Retained display library members are recovered from the support andanalyzed. The analysis can include amplification and a subsequentselection under similar or dissimilar conditions. For example, positiveand negative selections can be alternated. The analysis can also includedetermining the amino acid sequence of the polypeptide component, i.e.,the anti-mesothelin binding domain, and purification of the polypeptidecomponent for detailed characterization.

A variety of formats can be used for display libraries. Examples includethe phage display. In phage display, the protein component is typicallycovalently linked to a bacteriophage coat protein. The linkage resultsfrom translation of a nucleic acid encoding the protein component fusedto the coat protein. The linkage can include a flexible peptide linker,a protease site, or an amino acid incorporated as a result ofsuppression of a stop codon. Phage display is described, for example, inU.S. Pat. No. 5,223,409; Smith (1985) Science 228:1315-1317; WO92/18619; WO 91/17271; WO 92/20791; WO 92/15679; WO 93/01288; WO92/01047; WO 92/09690; WO 90/02809; de Haard et al. (1999) J. Biol. Chem274:18218-30; Hoogenboom et al. (1998) Immunotechnology 4:1-20;Hoogenboom et al. (2000) Immunol Today 2:371-8 and Hoet et al. (2005)Nat Biotechnol. 23(3) 344-8. Bacteriophage displaying the proteincomponent can be grown and harvested using standard phage preparatorymethods, e.g. PEG precipitation from growth media. After selection ofindividual display phages, the nucleic acid encoding the selectedprotein components can be isolated from cells infected with the selectedphages or from the phage themselves, after amplification. Individualcolonies or plaques can be picked, the nucleic acid isolated andsequenced.

Other display formats include cell based display (see, e.g., WO03/029456), protein-nucleic acid fusions (see, e.g., U.S. Pat. No.6,207,446), ribosome display (See, e.g., Mattheakis et al. (1994) Proc.Natl. Acad. Sci. USA 91:9022 and Hanes et al. (2000) Nat Biotechnol.18:1287-92; Hanes et al. (2000) Methods Enzymol. 328:404-30; andSchaffitzel et al. (1999) J Immunol Methods. 231(1-2):119-35), and E.coli periplasmic display (J Immunol Methods. 2005 Nov. 22; PMID:16337958).

In addition to the use of display libraries, other methods can be usedto obtain an anti-mesothelin binding domain. For example, mesothelin ora fragment thereof can be used as an antigen in a non-human animal,e.g., a rodent.

In one embodiment, the non-human animal includes at least a part of ahuman immunoglobulin gene. For example, it is possible to engineer mousestrains deficient in mouse antibody production with large fragments ofthe human Ig loci. Using the hybridoma technology, antigen-specificmonoclonal antibodies (Mabs) derived from the genes with the desiredspecificity may be produced and selected. See, e.g., XENOMOUSE™, Greenet al., 1994, Nat. Gen. 7:13-21; U.S. 2003-0070185, WO 96/34096,published Oct. 31, 1996, and PCT Application No. PCT/US96/05928, filedApr. 29, 1996.

In some instances, scFvs can be prepared according to method known inthe art (see, for example, Bird et al., (1988) Science 242:423-426 andHuston et al., (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883). ScFvmolecules can be produced by linking VH and VL regions together, e.g.,using flexible polypeptide linkers. The scFv molecules can comprise alinker (e.g., a Ser-Gly linker) with an optimized length and/or aminoacid composition. The linker length can greatly affect how the variableregions of an scFv fold and interact. In fact, if a short polypeptidelinker is employed (e.g., between 5-10 amino acids, intrachain foldingis prevented. Interchain folding is also required to bring the twovariable regions together to form a functional epitope binding site. Forexamples of linker orientation and size see, e.g., Hollinger et al. 1993Proc Natl Acad. Sci. U.S.A. 90:6444-6448, U.S. Patent ApplicationPublication Nos. 2005/0100543, 2005/0175606, 2007/0014794, and PCTpublication Nos. WO2006/020258 and WO2007/024715, is incorporated hereinby reference.

An scFv can comprise a linker of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, or moreamino acid residues between its VL and VH regions. The linker sequencemay comprise any naturally occurring amino acid. In some embodiments,the linker sequence comprises amino acids glycine and serine. In anotherembodiment, the linker sequence comprises sets of glycine and serinerepeats such as (Gly₄Ser)_(n), where n is a positive integer equal to orgreater than 1. (SEQ ID NO: 135) In one embodiment, the linker can be(Gly₄Ser)₄ (SEQ ID NO: 27) or (Gly₄Ser)₃ (SEQ ID NO: 28). Variation inthe linker length may retain or enhance activity, giving rise tosuperior efficacy in activity studies.

Stability and Mutations

The stability of an anti-mesothelin binding domain, e.g., scFv molecules(e.g., soluble scFv), can be evaluated in reference to the biophysicalproperties (e.g., thermal stability) of a conventional control scFvmolecule or a full length antibody. In one embodiment, the human scFvhas a thermal stability that is greater than about 0.1, about 0.25,about 0.5, about 0.75, about 1, about 1.25, about 1.5, about 1.75, about2, about 2.5, about 3, about 3.5, about 4, about 4.5, about 5, about5.5, about 6, about 6.5, about 7, about 7.5, about 8, about 8.5, about9, about 9.5, about 10 degrees, about 11 degrees, about 12 degrees,about 13 degrees, about 14 degrees, or about 15 degrees Celsius than acontrol binding molecule (e.g. a conventional scFv molecule) in thedescribed assays.

The improved thermal stability of the anti-mesothelin binding domain,e.g., scFv, is subsequently conferred to the entire mesothelin CARconstruct, leading to improved therapeutic properties of the mesothelinCAR construct. The thermal stability of the anti-mesothelin bindingdomain, e.g., scFv, can be improved by at least about 2° C. or 3° C. ascompared to a conventional antibody. In one embodiment, theanti-mesothelin binding domain, e.g., scFv, has a 1° C. improved thermalstability as compared to a conventional antibody. In another embodiment,the anti-mesothelin binding domain, e.g., scFv, has a 2° C. improvedthermal stability as compared to a conventional antibody. In anotherembodiment, the anti-mesothelin binding domain, e.g., scFv, has a 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15° C. improved thermal stability ascompared to a conventional antibody. Comparisons can be made, forexample, between the scFv molecules disclosed herein and scFv moleculesor Fab fragments of an antibody from which the scFv VH and VL werederived. Thermal stability can be measured using methods known in theart. For example, in one embodiment, Tm can be measured. Methods formeasuring Tm and other methods of determining protein stability aredescribed in more detail below.

Mutations in scFv (arising through direct mutagenesis of the solublescFv) alter the stability of the scFv and improve the overall stabilityof the scFv and the CART construct. Stability of the humanized scFv iscompared against the murine scFv using measurements such as Tm,temperature denaturation and temperature aggregation.

In one embodiment, the anti-mesothelin binding domain, e.g., scFv,comprises at least one mutation such that the mutated anti-mesothelinbinding domain, e.g., scFv, confers improved stability to theanti-mesothelin construct. In another embodiment, the anti-mesothelinbinding domain, e.g., scFv, comprises at least 1, 2, 3, 4, 5, 6, 7, 8,9, 10 mutations such that the mutated anti-mesothelin binding domain,e.g., scFv, confers improved stability to the anti-mesothelin construct.The binding capacity of the mutant scFvs can be determined using assaysdescribed in the Examples.

Binding Affinity

A wide variety of methods for determining binding affinity are known inthe art. An exemplary method for determining binding affinity employssurface plasmon resonance. Surface plasmon resonance is an opticalphenomenon that allows for the analysis of real-time biospecificinteractions by detection of alterations in protein concentrationswithin a biosensor matrix, for example using the BIAcore system(Pharmacia Biosensor AB, Uppsala, Sweden and Piscataway, N.J.). Forfurther descriptions, see Jonsson, U., et al. (1993) Ann. Biol. Clin.51:19-26; Jonsson, U., i (1991) Biotechniques 11:620-627; Johnsson, B.,et al. (1995) J. Mol. Recognit. 8:125-131; and Johnnson, B., et al.(1991) Anal. Biochem. 198:268-277.

In one aspect, the portion of a CAR composition of the inventioncomprising an antibody or fragment thereof comprises amino acidsequences that are homologous to the amino acid sequences describedherein, and wherein the antibody or fragment thereof retains the desiredfunctional properties of the anti-mesothelin antibody fragments of theinvention. In one specific aspect, the CAR composition of the inventioncomprises an antibody fragment. In a further aspect, that antibodyfragment comprises an scFv.

In various aspects, the portion comprising an antibody or antibodyfragment of the CAR composition of the invention is engineered bymodifying one or more amino acids within one or both variable regions(i.e., VH and/or VL), for example within one or more CDR regions and/orwithin one or more framework regions. In one specific aspect, the CARcomposition of the invention comprises an antibody fragment. In afurther aspect, that antibody fragment comprises an scFv.

It will be understood by one of ordinary skill in the art that theantibody or antibody fragment of the invention may further be modifiedsuch that they vary in amino acid sequence (e.g., from wild-type), butnot in desired activity. For example, additional nucleotidesubstitutions leading to amino acid substitutions at “non-essential”amino acid residues may be made to the protein. For example, anonessential amino acid residue in a molecule may be replaced withanother amino acid residue from the same side chain family. In anotherembodiment, a string of amino acids can be replaced with a structurallysimilar string that differs in order and/or composition of side chainfamily members, i.e., a conservative substitution, in which an aminoacid residue is replaced with an amino acid residue having a similarside chain, may be made.

Families of amino acid residues having similar side chains have beendefined in the art, including basic side chains (e.g., lysine, arginine,histidine), acidic side chains (e.g., aspartic acid, glutamic acid),uncharged polar side chains (e.g., glycine, asparagine, glutamine,serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g.,alanine, valine, leucine, isoleucine, proline, phenylalanine,methionine, tryptophan), beta-branched side chains (e.g., threonine,valine, isoleucine) and aromatic side chains (e.g., tyrosine,phenylalanine, tryptophan, histidine).

Percent identity in the context of two or more nucleic acids orpolypeptide sequences, refers to two or more sequences that are thesame. Two sequences are “substantially identical” if two sequences havea specified percentage of amino acid residues or nucleotides that arethe same (i.e., 60% identity, optionally 70%, 71%. 72%. 73%, 74%, 75%,76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity over aspecified region, or, when not specified, over the entire sequence),when compared and aligned for maximum correspondence over a comparisonwindow, or designated region as measured using one of the followingsequence comparison algorithms or by manual alignment and visualinspection. Optionally, the identity exists over a region that is atleast about 50 nucleotides (or 10 amino acids) in length, or morepreferably over a region that is 100 to 500 or 1000 or more nucleotides(or 20, 50, 200 or more amino acids) in length.

For sequence comparison, typically one sequence acts as a referencesequence, to which test sequences are compared. When using a sequencecomparison algorithm, test and reference sequences are entered into acomputer, subsequence coordinates are designated, if necessary, andsequence algorithm program parameters are designated. Default programparameters can be used, or alternative parameters can be designated. Thesequence comparison algorithm then calculates the percent sequenceidentities for the test sequences relative to the reference sequence,based on the program parameters. Methods of alignment of sequences forcomparison are well known in the art. Optimal alignment of sequences forcomparison can be conducted, e.g., by the local homology algorithm ofSmith and Waterman, (1970) Adv. Appl. Math. 2:482c, by the homologyalignment algorithm of Needleman and Wunsch, (1970) J. Mol. Biol.48:443, by the search for similarity method of Pearson and Lipman,(1988) Proc. Nat'l. Acad. Sci. USA 85:2444, by computerizedimplementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA inthe Wisconsin Genetics Software Package, Genetics Computer Group, 575Science Dr., Madison, Wis.), or by manual alignment and visualinspection (see, e.g., Brent et al., (2003) Current Protocols inMolecular Biology).

Two examples of algorithms that are suitable for determining percentsequence identity and sequence similarity are the BLAST and BLAST 2.0algorithms, which are described in Altschul et al., (1977) Nuc. AcidsRes. 25:3389-3402; and Altschul et al., (1990) J. Mol. Biol.215:403-410, respectively. Software for performing BLAST analyses ispublicly available through the National Center for BiotechnologyInformation.

The percent identity between two amino acid sequences can also bedetermined using the algorithm of E. Meyers and W. Miller, (1988)Comput. Appl. Biosci. 4:11-17) which has been incorporated into theALIGN program (version 2.0), using a PAM120 weight residue table, a gaplength penalty of 12 and a gap penalty of 4. In addition, the percentidentity between two amino acid sequences can be determined using theNeedleman and Wunsch (1970) J. Mol. Biol. 48:444-453) algorithm whichhas been incorporated into the GAP program in the GCG software package(available at www.gcg.com), using either a Blossom 62 matrix or a PAM250matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a lengthweight of 1, 2, 3, 4, 5, or 6.

In one aspect, the present invention contemplates modifications of thestarting antibody or fragment (e.g., scFv) amino acid sequence thatgenerate functionally equivalent molecules. For example, the VH or VL ofan anti-mesothelin binding domain, e.g., scFv comprised in the CAR canbe modified to retain at least about 70%, 71%. 72%. 73%, 74%, 75%, 76%,77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity of the starting VHor VL framework region of the anti-mesothelin binding domain, e.g.,scFv. The present invention contemplates modifications of the entire CARconstruct, e.g., modifications in one or more amino acid sequences ofthe various domains of the CAR construct in order to generatefunctionally equivalent molecules. The CAR construct can be modified toretain at least about 70%, 71%. 72%. 73%, 74%, 75%, 76%, 77%, 78%, 79%,80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99% identity of the starting CAR construct.

Transmembrane Domain

With respect to the transmembrane domain, in various embodiments, a CARcan be designed to comprise a transmembrane domain that is attached tothe extracellular domain of the CAR. A transmembrane domain can includeone or more additional amino acids adjacent to the transmembrane region,e.g., one or more amino acid associated with the extracellular region ofthe protein from which the transmembrane was derived (e.g., 1, 2, 3, 4,5, 6, 7, 8, 9, 10 up to 15 amino acids of the extracellular region)and/or one or more additional amino acids associated with theintracellular region of the protein from which the transmembrane proteinis derived (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 up to 15 amino acids ofthe intracellular region). In one aspect, the transmembrane domain isone that is associated with one of the other domains of the CAR is used,e.g., in one embodiment, the transmembrane domain may be from the sameprotein that the signaling domain, costimulatory domain or the hingedomain is derived from. In another aspect, the transmembrane domain isnot derived from the same protein that any other domain of the CAR isderived from. In some instances, the transmembrane domain can beselected or modified by amino acid substitution to avoid binding of suchdomains to the transmembrane domains of the same or different surfacemembrane proteins, e.g., to minimize interactions with other members ofthe receptor complex. In one aspect, the transmembrane domain is capableof homodimerization with another CAR on the cell surface of aCAR-expressing cell. In a different aspect, the amino acid sequence ofthe transmembrane domain may be modified or substituted so as tominimize interactions with the binding domains of the native bindingpartner present in the same CAR-expressing cell.

The transmembrane domain may be derived either from a natural or from arecombinant source. Where the source is natural, the domain may bederived from any membrane-bound or transmembrane protein. In one aspect,the transmembrane domain is capable of signaling to the intracellulardomain(s) whenever the CAR has bound to a target. A transmembrane domainof particular use in this invention may include at least thetransmembrane domain(s) of, e.g., the alpha, beta or zeta chain of theT-cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8 (e.g., CD8alpha, CD8 beta), CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134,CD137, CD154. In some embodiments, a transmembrane domain may include atleast the transmembrane region(s) of, e.g., KIRDS2, OX40, CD2, CD27,LFA-1 (CD11a, CD18), ICOS (CD278), 4-1BB (CD137), GITR, CD40, BAFFR,HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD160, CD19,IL2R beta, IL2R gamma, IL7R a, ITGA1, VLA1, CD49a, ITGA4, IA4, CD49D,ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1,ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7,TNFR2, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile),CEACAM1, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), SLAMF6(NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG(CD162), LTBR, PAG/Cbp, NKG2D, and NKG2C.

In some instances, the transmembrane domain can be attached to theextracellular region of the CAR, e.g., the antigen binding domain of theCAR, via a hinge, e.g., a hinge from a human protein. For example, inone embodiment, the hinge can be a human Ig (immunoglobulin) hinge(e.g., an IgG4 hinge, an IgD hinge), a GS linker (e.g., a GS linkerdescribed herein), a KIR2DS2 hinge or a CD8a hinge. In one embodiment,the hinge or spacer comprises (e.g., consists of) the amino acidsequence of SEQ ID NO:2. In one aspect, the transmembrane domaincomprises (e.g., consists of) a transmembrane domain of SEQ ID NO: 6.

In one aspect, the hinge or spacer comprises an IgG4 hinge. For example,in one embodiment, the hinge or spacer comprises a hinge of the aminoacid sequence as follows:

(SEQ ID NO: 3) ESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGKM.

In some embodiments, the hinge or spacer comprises a hinge encoded by anucleotide sequence as follows:

(SEQ ID NO: 14) GAGAGCAAGTACGGCCCTCCCTGCCCCCCTTGCCCTGCCCCCGAGTTCCTGGGCGGACCCAGCGTGTTCCTGTTCCCCCCCAAGCCCAAGGACACCCTGATGATCAGCCGGACCCCCGAGGTGACCTGTGTGGTGGTGGACGTGTCCCAGGAGGACCCCGAGGTCCAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCACAACGCCAAGACCAAGCCCCGGGAGGAGCAGTTCAATAGCACCTACCGGGTGGTGTCCGTGCTGACCGTGCTGCACCAGGACTGGCTGAACGGCAAGGAATACAAGTGTAAGGTGTCCAACAAGGGCCTGCCCAGCAGCATCGAGAAAACCATCAGCAAGGCCAAGGGCCAGCCTCGGGAGCCCCAGGTGTACACCCTGCCCCCTAGCCAAGAGGAGATGACCAAGAACCAGGTGTCCCTGACCTGCCTGGTGAAGGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAACGGCCAGCCCGAGAACAACTACAAGACCACCCCCCCTGTGCTGGACAGCGACGGCAGCTTCTTCCTGTACAGCCGGCTGACCGTGGACAAGAGCCGGTGGCAGGAGGGCAACGTCTTTAGCTGCTCCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGAGCCTGAGCCTGTCCCTGGGCAAGATG. 

In one aspect, the hinge or spacer comprises an IgD hinge. For example,in one embodiment, the hinge or spacer comprises a hinge of the aminoacid sequence

(SEQ ID NO: 4) RWPESPKAQASSVPTAQPQAEGSLAKATTAPATTRNTGRGGEEKKKEKEKEEQEERETKTPECPSHTQPLGVYLLTPAVQDLWLRDKATFTCFVVGSDLKDAHLTWEVAGKVPTGGVEEGLLERHSNGSQSQHSRLTLPRSLWNAGTSVTCTLNHPSLPPQRLMALREPAAQAPVKLSLNLLASSDPPEAASWLLCEVSGFSPPNILLMWLEDQREVNTSGEAPARPPPQPGSTTFWAWSVLRVPAPPSPQPATYTCVVSHEDSRTLLNASRSLEVSYVTDH. 

In some embodiments, the hinge or spacer comprises a hinge encoded by anucleotide sequence of

(SEQ ID NO: 15) AGGTGGCCCGAAAGTCCCAAGGCCCAGGCATCTAGTGTTCCTACTGCACAGCCCCAGGCAGAAGGCAGCCTAGCCAAAGCTACTACTGCACCTGCCACTACGCGCAATACTGGCCGTGGCGGGGAGGAGAAGAAAAAGGAGAAAGAGAAAGAAGAACAGGAAGAGAGGGAGACCAAGACCCCTGAATGTCCATCCCATACCCAGCCGCTGGGCGTCTATCTCTTGACTCCCGCAGTACAGGACTTGTGGCTTAGAGATAAGGCCACCTTTACATGTTTCGTCGTGGGCTCTGACCTGAAGGATGCCCATTTGACTTGGGAGGTTGCCGGAAAGGTACCCACAGGGGGGGTTGAGGAAGGGTTGCTGGAGCGCCATTCCAATGGCTCTCAGAGCCAGCACTCAAGACTCACCCTTCCGAGATCCCTGTGGAACGCCGGGACCTCTGTCACATGTACTCTAAATCATCCTAGCCTGCCCCCACAGCGTCTGATGGCCCTTAGAGAGCCAGCCGCCCAGGCACCAGTTAAGCTTAGCCTGAATCTGCTCGCCAGTAGTGATCCCCCAGAGGCCGCCAGCTGGCTCTTATGCGAAGTGTCCGGCTTTAGCCCGCCCAACATCTTGCTCATGTGGCTGGAGGACCAGCGAGAAGTGAACACCAGCGGCTTCGCTCCAGCCCGGCCCCCACCCCAGCCGGGTTCTACCACATTCTGGGCCTGGAGTGTCTTAAGGGTCCCAGCACCACCTAGCCCCCAGCCAGCCACATACACCTGTGTTGTGTCCCATGAAGATAGCAGGACCCTGCTAAATGCTTCTAGGAGTCTGGAGGTTTCCTACGTGACTGACCATT.

In one aspect, the transmembrane domain may be recombinant, in whichcase it will comprise predominantly hydrophobic residues such as leucineand valine. In one aspect a triplet of phenylalanine, tryptophan andvaline can be found at each end of a recombinant transmembrane domain.

Optionally, a short oligo- or polypeptide linker, between 2 and 10 aminoacids in length may form the linkage between the transmembrane domainand the cytoplasmic signaling region of the CAR. A glycine-serinedoublet provides a particularly suitable linker. For example, in oneaspect, the linker comprises the amino acid sequence of GGGGSGGGGS (SEQID NO:5). In some embodiments, the linker is encoded by a nucleotidesequence of

(SEQ ID NO: 16) GGTGGCGGAGGTTCTGGAGGTGGAGGTTCC.

In one aspect, the hinge or spacer comprises a KIR2DS2 hinge andportions thereof.

Cytoplasmic Domain

The cytoplasmic domain or region of the CAR includes an intracellularsignaling domain. An intracellular signaling domain is generallyresponsible for activation of at least one of the normal effectorfunctions of the immune cell in which the CAR has been introducede. Theterm “effector function” refers to a specialized function of a cell.Effector function of a T cell, for example, may be cytolytic activity orhelper activity including the secretion of cytokines. Thus the term“intracellular signaling domain” refers to the portion of a proteinwhich transduces the effector function signal and directs the cell toperform a specialized function. While usually the entire intracellularsignaling domain can be employed, in many cases it is not necessary touse the entire chain. To the extent that a truncated portion of theintracellular signaling domain is used, such truncated portion may beused in place of the intact chain as long as it transduces the effectorfunction signal. The term intracellular signaling domain is thus meantto include any truncated portion of the intracellular signaling domainsufficient to transduce the effector function signal.

Examples of intracellular signaling domains for use in the CAR of theinvention include the cytoplasmic sequences of the T cell receptor (TCR)and co-receptors that act in concert to initiate signal transductionfollowing antigen receptor engagement, as well as any derivative orvariant of these sequences and any recombinant sequence that has thesame functional capability.

It is known that signals generated through the TCR alone areinsufficient for full activation of the T cell and that a secondaryand/or costimulatory signal is also required. Thus, T cell activationcan be said to be mediated by two distinct classes of cytoplasmicsignaling sequences: those that initiate antigen-dependent primaryactivation through the TCR (primary intracellular signaling domains) andthose that act in an antigen-independent manner to provide a secondaryor costimulatory signal (secondary cytoplasmic domain, e.g., acostimulatory domain).

A primary cytoplasmic signaling domain regulates primary activation ofthe TCR complex either in a stimulatory way, or in an inhibitory way.Primary intracellular signaling domains that act in a stimulatory mannermay contain signaling motifs which are known as immunoreceptortyrosine-based activation motifs or ITAMs.

Examples of ITAM containing primary intracellular signaling domains thatare of particular use in the invention include those of CD3 zeta, commonFcR gamma (FCER1G), Fc gamma RIIa, FcR beta (Fc Epsilon Rib), CD3 gamma,CD3 delta, CD3 epsilon, CD79a, CD79b, DAP10, and DAP12. In oneembodiment, a CAR of the invention comprises an intracellular signalingdomain, e.g., a primary signaling domain of CD3-zeta.

In one embodiment, a primary signaling domain comprises a modified ITAMdomain, e.g., a mutated ITAM domain which has altered (e.g., increasedor decreased) activity as compared to the native ITAM domain. In oneembodiment, a primary signaling domain comprises a modifiedITAM-containing primary intracellular signaling domain, e.g., anoptimized and/or truncated ITAM-containing primary intracellularsignaling domain. In an embodiment, a primary signaling domain comprisesone, two, three, four or more ITAM motifs.

Further examples of molecules containing a primary intracellularsignaling domain that are of particular use in the invention includethose of DAP10, DAP12, and CD32.

The intracellular domain of the CAR can comprise the CD3-zeta signalingdomain by itself or it can be combined with any other desiredintracellular signaling domain(s) useful in the context of a CAR of theinvention. For example, the intracellular signaling domain of the CARcan comprise a CD3 zeta chain portion and a costimulatory signalingdomain. The costimulatory signaling domain refers to a portion of theCAR comprising the intracellular domain of a costimulatory molecule. Acostimulatory molecule is a cell surface molecule other than an antigenreceptor or its ligands that is required for an efficient response oflymphocytes to an antigen. Examples of such molecules include CD27,CD28, 4-1BB (CD137), OX40, CD30, CD40, PD-1 (also known as PD1), ICOS,lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT,NKG2C, B7-H3, and a ligand that specifically binds with CD83, and thelike. For example, CD27 costimulation has been demonstrated to enhanceexpansion, effector function, and survival of human CART cells in vitroand augments human T cell persistence and antitumor activity in vivo(Song et al. Blood. 2012; 119(3):696-706). Further examples of suchcostimulatory molecules include CD5, ICAM-1, GITR, BAFFR, HVEM (LIGHTR),SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD160, CD19, CD4, CD8alpha,CD8beta, IL2R beta, IL2R gamma, IL7R alpha, ITGA4, VLA1, CD49a, ITGA4,IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL,CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18,LFA-1, ITGB7, TNFR2, TRANCE/RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4),CD84, CD96 (Tactile), NKG2D, CEACAM1, CRTAM, Ly9 (CD229), CD160 (BY55),PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150,IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, LAT, GADS, SLP-76, andPAG/Cbp.

The intracellular signaling domains within the cytoplasmic portion ofthe CAR of the invention may be linked to each other in a random orspecified order. Optionally, a short oligo- or polypeptide linker, forexample, between 2 and 10 amino acids (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or10 amino acids) in length may form the linkage between intracellularsignaling domains. In one embodiment, a glycine-serine doublet can beused as a suitable linker. In one embodiment, a single amino acid, e.g.,an alanine, a glycine, can be used as a suitable linker.

In one aspect, the intracellular signaling domain is designed tocomprise two or more, e.g., 2, 3, 4, 5, or more, costimulatory signalingdomains. In an embodiment, the two or more, e.g., 2, 3, 4, 5, or more,costimulatory signaling domains, are separated by a linker molecule,e.g., a linker molecule described herein. In one embodiment, theintracellular signaling domain comprises two costimulatory signalingdomains. In some embodiments, the linker molecule is a glycine residue.In some embodiments, the linker is an alanine residue.

In one aspect, the intracellular signaling domain is designed tocomprise the signaling domain of CD3-zeta and the signaling domain ofCD28. In one aspect, the intracellular signaling domain is designed tocomprise the signaling domain of CD3-zeta and the signaling domain of4-1BB. In one aspect, the signaling domain of 4-1BB is a signalingdomain of SEQ ID NO: 16. In one aspect, the signaling domain of CD3-zetais a signaling domain of SEQ ID NO: 17.

In one aspect, the intracellular signaling domain is designed tocomprise the signaling domain of CD3-zeta and the signaling domain ofCD27. In one aspect, the signaling domain of CD27 comprises an aminoacid sequence of

(SEQ ID NO: 8) QRRKYRSNKGESPVEPAEPCRYSCPREEEGSTIPIQEDYRKPEPACSP.In one aspect, the signalling domain of CD27 is encoded by a nucleicacid sequence of

(SEQ ID NO: 19) AGGAGTAAGAGGAGCAGGCTCCTGCACAGTGACTACATGAACATGACTCCCCGCCGCCCCGGGCCCACCCGCAAGCATTACCAGCCCTATGCCCCACCACGCGACTTCGCAGCCTATCGCTCC.

In one aspect, the CAR-expressing cell described herein can furthercomprise a second CAR, e.g., a second CAR that includes a differentantigen binding domain, e.g., to the same target (mesothelin) or adifferent target (e.g., a target other than mesothelin on stroma cells,e.g., FAP; a target other than mesothelin on prostate cancer cells,e.g., androgen receptor, OR51E2, PSMA, PSCA, PDGRF-β, TARP, GloboH,MAD-CT-1, or MAD-CT-2; a target other than mesothelin on ovarariancancer cells, e.g., Tn, PRSS21, CD171, Lewis Y, folate receptor α,claudin6, GloboH, or sperm protein 17, e.g., a target other thanmesothelin on lung cancer cells, e.g., VEGF, HER3, IGF-1R, EGFR, DLL4,or Trop-2). In one embodiment, the CAR-expressing cell comprises a firstCAR that targets a first antigen and includes an intracellular signalingdomain having a costimulatory signaling domain but not a primarysignaling domain, and a second CAR that targets a second, different,antigen and includes an intracellular signaling domain having a primarysignaling domain but not a costimulatory signaling domain. Placement ofa costimulatory signaling domain, e.g., 4-1BB, CD28, CD27 or OX-40, ontothe first CAR, and the primary signaling domain, e.g., CD3 zeta, on thesecond CAR can limit the CAR activity to cells where both targets areexpressed. In one embodiment, the CAR expressing cell comprises a firstmesothelin CAR that includes a mesothelin binding domain, atransmembrane domain and a costimulatory domain and a second CAR thattargets an antigen other than mesothelin (e.g., a target other thanmesothelin on stroma cells, e.g., FAP; a target other than mesothelin onprostate cancer cells, e.g., androgen receptor, OR51E2, PSMA, PSCA,PDGRF-β, TARP, GloboH, MAD-CT-1, or MAD-CT-2; a target other thanmesothelin on ovararian cancer cells, e.g., Tn, PRSS21, CD171, Lewis Y,folate receptor α, claudin6, GloboH, or sperm protein 17, e.g., a targetother than mesothelin on lung cancer cells, e.g., VEGF, HER3, IGF-1R,EGFR, DLL4, or Trop-2) and includes an antigen binding domain, atransmembrane domain and a primary signaling domain. In anotherembodiment, the CAR expressing cell comprises a first mesothelin CARthat includes a mesothelin binding domain, a transmembrane domain and aprimary signaling domain and a second CAR that targets an antigen otherthan mesothelin (e.g., a target other than mesothelin on stroma cells,e.g., FAP; a target other than mesothelin on prostate cancer cells,e.g., androgen receptor, OR51E2, PSMA, PSCA, PDGRF-β, TARP, GloboH,MAD-CT-1, or MAD-CT-2; a target other than mesothelin on ovarariancancer cells, e.g., Tn, PRSS21, CD171, Lewis Y, folate receptor α,claudin6, GloboH, or sperm protein 17, e.g., a target other thanmesothelin on lung cancer cells, e.g., VEGF, HER3, IGF-1R, EGFR, DLL4,or Trop-2) and includes an antigen binding domain to the antigen, atransmembrane domain and a costimulatory signaling domain.

In one embodiment, the CAR-expressing cell comprises a mesothelin CARdescribed herein and an inhibitory CAR. In one embodiment, theinhibitory CAR comprises an antigen binding domain that binds an antigenfound on normal cells but not cancer cells, e.g., normal cells that alsoexpress mesothelin. In one embodiment, the inhibitory CAR comprises theantigen binding domain, a transmembrane domain and an intracellulardomain of an inhibitory molecule. For example, the intracellular domainof the inhibitory CAR can be an intracellular domain of PD1, PD-L1,CTLA4, TIM3, CEACAM (e.g., CEACAM-1, CEACAM-3 and/or CEACAM-5), LAG3,VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4 and TGFR beta.

In one embodiment, when the CAR-expressing cell comprises two or moredifferent CARs, the antigen binding domains of the different CARs can besuch that the antigen binding domains do not interact with one another.For example, a cell expressing a first and second CAR can have anantigen binding domain of the first CAR, e.g., as a fragment, e.g., anscFv, that does not form an association with the antigen binding domainof the second CAR, e.g., the antigen binding domain of the second CAR isa VHH.

In some embodiments, the antigen binding domain comprises a singledomain antigen binding (SDAB) molecules include molecules whosecomplementary determining regions are part of a single domainpolypeptide. Examples include, but are not limited to, heavy chainvariable domains, binding molecules naturally devoid of light chains,single domains derived from conventional 4-chain antibodies, engineereddomains and single domain scaffolds other than those derived fromantibodies. SDAB molecules may be any of the art, or any future singledomain molecules. SDAB molecules may be derived from any speciesincluding, but not limited to mouse, human, camel, llama, lamprey, fish,shark, goat, rabbit, and bovine. This term also includes naturallyoccurring single domain antibody molecules from species other thanCamelidae and sharks.

In one aspect, an SDAB molecule can be derived from a variable region ofthe immunoglobulin found in fish, such as, for example, that which isderived from the immunoglobulin isotype known as Novel Antigen Receptor(NAR) found in the serum of shark. Methods of producing single domainmolecules derived from a variable region of NAR (“IgNARs”) are describedin WO 03/014161 and Streltsov (2005) Protein Sci. 14:2901-2909.

According to another aspect, an SDAB molecule is a naturally occurringsingle domain antigen binding molecule known as heavy chain devoid oflight chains. Such single domain molecules are disclosed in WO 9404678and Hamers-Casterman, C. et al. (1993) Nature 363:446-448, for example.For clarity reasons, this variable domain derived from a heavy chainmolecule naturally devoid of light chain is known herein as a VHH ornanobody to distinguish it from the conventional VH of four chainimmunoglobulins. Such a VHH molecule can be derived from Camelidaespecies, for example in camel, llama, dromedary, alpaca and guanaco.Other species besides Camelidae may produce heavy chain moleculesnaturally devoid of light chain; such VHHs are within the scope of theinvention.

The SDAB molecules can be recombinant, CDR-grafted, humanized,camelized, de-immunized and/or in vitro generated (e.g., selected byphage display).

It has also been discovered, that cells having a plurality of chimericmembrane embedded receptors comprising an antigen binding domain thatinteractions between the antigen binding domain of the receptors can beundesirable, e.g., because it inhibits the ability of one or more of theantigen binding domains to bind its cognate antigen. Accordingly,disclosed herein are cells having a first and a second non-naturallyoccurring chimeric membrane embedded receptor comprising antigen bindingdomains that minimize such interactions. Also disclosed herein arenucleic acids encoding a first and a second non-naturally occurringchimeric membrane embedded receptor comprising an antigen bindingdomains that minimize such interactions, as well as methods of makingand using such cells and nucleic acids. In an embodiment the antigenbinding domain of one of the first and the second non-naturallyoccurring chimeric membrane embedded receptor, comprises an scFv, andthe other comprises a single VH domain, e.g., a camelid, shark, orlamprey single VH domain, or a single VH domain derived from a human ormouse sequence.

In some embodiments, the claimed invention comprises a first and secondCAR, wherein the antigen binding domain of one of the first and thesecond CAR does not comprise a variable light domain and a variableheavy domain. In some embodiments, the antigen binding domain of one ofthe first and the second CAR is an scFv, and the other is not an scFv.In some embodiments, the antigen binding domain of one of the first andthe second CAR comprises a single VH domain, e.g., a camelid, shark, orlamprey single VH domain, or a single VH domain derived from a human ormouse sequence. In some embodiments, the antigen binding domain of oneof the first and the second CAR comprises a nanobody. In someembodiments, the antigen binding domain of one of the first and thesecond CAR comprises a camelid VHH domain.

In some embodiments, the antigen binding domain of one of the first andthesecond CAR comprises an scFv, and the other comprises a single VHdomain, e.g., a camelid, shark, or lamprey single VH domain, or a singleVH domain derived from a human or mouse sequence. In some embodiments,the antigen binding domain of one of the first and the second CARcomprises an scFv, and the other comprises a nanobody. In someembodiments, the antigen binding domain of one of the first and thesecond CAR comprises comprises an scFv, and the other comprises acamelid VHH domain.

In some embodiments, when present on the surface of a cell, binding ofthe antigen binding domain of the first CAR to its cognate antigen isnot substantially reduced by the presence of the second CAR. In someembodiments, binding of the antigen binding domain of the first CAR toits cognate antigen in the presence of the second CAR is 85%, 90%, 95%,96%, 97%, 98% or 99% of binding of the antigen binding domain of thefirst CAR to its cognate antigen in the absence of the second CAR.

In some embodiments, when present on the surface of a cell, the antigenbinding domains of the first and the second CAR, associate with oneanother less than if both were scFv antigen binding domains. In someembodiments, the antigen binding domains of the first and the secondCAR, associate with one another 85%, 90%, 95%, 96%, 97%, 98% or 99% lessthan if both were scFv antigen binding domains.

In another aspect, the CAR-expressing cell described herein can furtherexpress another agent, e.g., an agent which enhances the activity orfitness of a CAR-expressing cell. For example, in one embodiment, theagent can be an agent which inhibits a molecule that modulates orregulates, e.g., inhibits, T cell function. In some embodiments, themolecule that modulates or regulates T cell function is an inhibitorymolecule. Inhibitory molecules, e.g., PD1, can, in some embodiments,decrease the ability of a CAR-expressing cell to mount an immuneeffector response. Examples of inhibitory molecules include PD1, PD-L1,CTLA4, TIM3, CEACAM (e.g., CEACAM-1, CEACAM-3 and/or CEACAM-5), LAG3,VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4 and TGFR beta. In embodiments, anagent, e.g., an inhibitory nucleic acid, e.g., a dsRNA, e.g., an siRNAor shRNA; or e.g., an inhibitory protein or system, e.g., a clusteredregularly interspaced short palindromic repeats (CRISPR), atranscription-activator like effector nuclease (TALEN), or a zinc fingerendonuclease (ZFN), e.g., as described herein, can be used to inhibitexpression of a molecule that modulates or regulates, e.g., inhibits,T-cell function in the CAR-expressing cell. In an embodiment the agentis an shRNA, e.g., an shRNA described herein. In an embodiment, theagent that modulates or regulates, e.g., inhibits, T-cell function isinhibited within a CAR-expressing cell. For example, a dsRNA moleculethat inhibits expression of a molecule that modulates or regulates,e.g., inhibits, T-cell function is linked to the nucleic acid thatencodes a component, e.g., all of the components, of the CAR.

In one embodiment, the agent which inhibits an inhibitory moleculecomprises a first polypeptide, e.g., an inhibitory molecule, associatedwith a second polypeptide that provides a positive signal to the cell,e.g., an intracellular signaling domain described herein. In oneembodiment, the agent comprises a first polypeptide, e.g., of aninhibitory molecule such as PD1, PD-L1, CTLA4, TIM3, CEACAM (e.g.,CEACAM-1, CEACAM-3 and/or CEACAM-5), LAG3, VISTA, BTLA, TIGIT, LAIR1,CD160, 2B4 and TGFR beta T, or a fragment of any of these (e.g., atleast a portion of an extracellular domain of any of these), and asecond polypeptide which is an intracellular signaling domain describedherein (e.g., comprising a costimulatory domain (e.g., 41BB, CD27 orCD28, e.g., as described herein) and/or a primary signaling domain(e.g., a CD3 zeta signaling domain described herein). In one embodiment,the agent comprises a first polypeptide of PD1 or a fragment thereof(e.g., at least a portion of an extracellular domain of PD1), and asecond polypeptide of an intracellular signaling domain described herein(e.g., a CD28 signaling domain described herein and/or a CD3 zetasignaling domain described herein). PD1 is an inhibitory member of theCD28 family of receptors that also includes CD28, CTLA-4, ICOS, andBTLA. PD-1 is expressed on activated B cells, T cells and myeloid cells(Agata et al. 1996 Int. Immunol 8:765-75). Two ligands for PD1, PD-L1and PD-L2 have been shown to downregulate T cell activation upon bindingto PD1 (Freeman et a. 2000 J Exp Med 192:1027-34; Latchman et al. 2001Nat Immunol 2:261-8; Carter et al. 2002 Eur J Immunol 32:634-43). PD-L1is abundant in human cancers (Dong et al. 2003 J Mol Med 81:281-7; Blanket al. 2005 Cancer Immunol. Immunother 54:307-314; Konishi et al. 2004Clin Cancer Res 10:5094). Immune suppression can be reversed byinhibiting the local interaction of PD1 with PD-L1.

In one embodiment, the agent comprises the extracellular domain (ECD) ofan inhibitory molecule, e.g., Programmed Death 1 (PD1), can be fused toa transmembrane domain and intracellular signaling domains such as 41BBand CD3 zeta (also referred to herein as a PD1 CAR). In one embodiment,the PD1 CAR, when used in combinations with a mesothelin CAR describedherein, improves the persistence of the T cell. In one embodiment, theCAR is a PD1 CAR comprising the extracellular domain of PD1 indicated asunderlined in SEQ ID NO: 24 and a signal sequence at amino acids 1-21 ofSEQ ID NO:24. In one embodiment, the PD1 CAR comprises the amino acidsequence of SEQ ID NO:24.

(SEQ ID NO: 24) Malpvtalllplalllhaarppgwfldspdrpwnpptfspallvvtegdnatftcsfsntsesfvlnwyrmspsnqtdklaafpedrsqpgqdcrfrvtqlpngrdfhmsvvrarrndsgtylcgaislapkaqikeslraelrvterraevptahpspsprpagqfqtlvtttpaprpptpaptiasqplslrpeacrpaaggavhtrgldfacdiyiwaplagtcgvlllslvitlyckrgrkkllyifkqpfmrpvqttqeedgcscrfpeeeeggcelrvkfsrsadapaykqgqnqlynelnlgrreeydvldkrrgrdpemggkprrknpqeglynelqkdkmaeayseigmkgerrrgkghdglyqglstatkdtydalhmqalppr.

In one embodiment, the PD1 CAR without the N-terminal signal sequencecomprises the amino acid sequence provided below (SEQ ID NO:22).

(SEQ ID NO: 22) pgwfldspdrpwnpptfspallvvtegdnatftcsfsntsesfvlnwyrmspsnqtdklaafpedrsqpgqdcrfrvtqlpngrdfhmsvvrarrndsgtylcgaislapkaqikeslraelrvterraevptahpspsprpagqfqtlvtttpaprpptpaptiasqplslrpeacrpaaggavhtrgldfacdiyiwaplagtcgvlllslvitlyckrgrkkllyifkqpfmrpvqttqeedgcscrfpeeeeggcelrvkfsrsadapaykqgqnqlynelnlgrreeydvldkrrgrdpemggkprrknpqeglynelqkdkmaeayseigmkgerrrgkghdglyqglstatkdtydalhmqalppr.

In one embodiment, the agent comprises a nucleic acid sequence encodingthe PD1 CAR with the N-terminal signal sequence, e.g., the PD1 CARdescribed herein. In one embodiment, the nucleic acid sequence for thePD1 CAR is shown below, with the PD1 ECD underlined below in SEQ ID NO:23

(SEQ ID NO: 23) atggccctccctgtcactgccctgcttctccccctcgcactcctgctccacgccgctagaccacccggatggtttctggactctccggatcgcccgtggaatcccccaaccttctcaccggcactcttggttgtgactgagggcgataatgcgaccttcacgtgctcgttctccaacacctccgaatcattcgtgctgaactggtaccgcatgagcccgtcaaaccagaccgacaagctcgccgcgtttccggaagatcggtcgcaaccgggacaggattgtcggttccgcgtgactcaactgccgaatggcagagacttccacatgagcgtggtccgcgctaggcgaaacgactccgggacctacctgtgcggagccatctcgctggcgcctaaggcccaaatcaaagagagcttgagggccgaactgagagtgaccgagcgcagagctgaggtgccaactgcacatccatccccatcgcctcggcctgcggggcagtttcagaccctggtcacgaccactccggcgccgcgcccaccgactccggccccaactatcgcgagccagcccctgtcgctgaggccggaagcatgccgccctgccgccggaggtgctgtgcatacccggggattggacttcgcatgcgacatctacatttgggctcctctcgccggaacttgtggcgtgctccttctgtccctggtcatcaccctgtactgcaagcggggtcggaaaaagatctgtacattttcaagcagccatcatgaggcccgtgcaaaccacccaggaggaggacggttgctcctgccggttccccgaagaggaagaaggaggttgcgagctgcgcgtgaagttctcccggagcgccgacgcccccgcctataagcagggccagaaccagctgtacaacgaactgaacctgggacggcgggaagagtacgatgtgctggacaagcggcgcggccgggaccccgaaatgggcgggaagcctagaagaaagaaccctcaggaaggcctgtataacgagctgcagaaggacaagatggccgaggcctactccgaaattgggatgaagggagagcggcggaggggaaaggggcacgacggcctgtaccaaggactgtccaccgccaccaaggacacatacgatgccctgcacatgcaggcccttccccctcgc.

In another aspect, the present invention provides a population ofCAR-expressing cells, e.g., CART cells. In some embodiments, thepopulation of CAR-expressing cells comprises a mixture of cellsexpressing different CARs. For example, in one embodiment, thepopulation of CART cells can include a first cell expressing a CARhaving an anti-CD19 binding domain described herein, and a second cellexpressing a CAR having a different anti-CD19 binding domain, e.g., ananti-mesothelin binding domain described herein that differs from theanti-mesothelin binding domain in the CAR expressed by the first cell.As another example, the population of CAR-expressing cells can include afirst cell expressing a CAR that includes an anti-mesothelin bindingdomain, e.g., as described herein, and a second cell expressing a CARthat includes an antigen binding domain to a target other thanmesothelin (e.g., a target other than mesothelin on stroma cells, e.g.,FAP; a target other than mesothelin on prostate cancer cells, e.g.,androgen receptor, 0101E2, PSMA, PSCA, PDGRF-β, TARP, GloboH, MAD-CT-1,or MAD-CT-2; a target other than mesothelin on ovararian cancer cells,e.g., Tn, PRSS21, CD171, Lewis Y, folate receptor α, claudin6, GloboH,or sperm protein 17, e.g., a target other than mesothelin on lung cancercells, e.g., VEGF, HER3, IGF-1R, EGFR, DLL4, or Trop-2). In oneembodiment, the population of CAR-expressing cells includes, e.g., afirst cell expressing a CAR that includes a primary intracellularsignaling domain, and a second cell expressing a CAR that includes asecondary signaling domain.

In another aspect, the present invention provides a population of cellswherein at least one cell in the population expresses a CAR having ananti-mesothelin binding domain described herein, and a second cellexpressing another agent, e.g., an agent which enhances the activity orfunction of a CAR-expressing cell. For example, in one embodiment, theagent can be an agent which modulates or regulates, e.g., inhibits, Tcell function. In some embodiments, the molecule that modulates orregulates T cell function is an inhibitory molecule, e.g., an agentdescribed herein. Inhibitory molecules, e.g., can, in some embodiments,decrease the ability of a CAR-expressing cell to mount an immuneeffector response. Examples of inhibitory molecules include PD1, PD-L1,CTLA4, TIM3, CEACAM (e.g., CEACAM-1, CEACAM-3 and/or CEACAM-5), LAG3,VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4 and TGFR beta. In one embodiment,the agent which inhibits an inhibitory molecule comprises a firstpolypeptide, e.g., an inhibitory molecule, associated with a secondpolypeptide that provides a positive signal to the cell, e.g., anintracellular signaling domain described herein. In one embodiment, theagent comprises a first polypeptide, e.g., of an inhibitory moleculesuch as PD1, PD-L1, CTLA4, TIM3, CEACAM (e.g., CEACAM-1, CEACAM-3 and/orCEACAM-5), LAG3, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4 and TGFR beta, ora fragment of any of these (e.g., at least a portion of an extracellulardomain of any of these), and a second polypeptide which is anintracellular signaling domain described herein (e.g., comprising acostimulatory domain (e.g., 41BB, CD27 or CD28, e.g., as describedherein) and/or a primary signaling domain (e.g., a CD3 zeta signalingdomain described herein). In one embodiment, the agent comprises a firstpolypeptide of PD1 or a fragment thereof (e.g., at least a portion ofthe extracellular domain of PD1), and a second polypeptide of anintracellular signaling domain described herein (e.g., a CD28 signalingdomain described herein and/or a CD3 zeta signaling domain describedherein).

In one aspect, the present invention provides methods comprisingadministering a population of CAR-expressing cells, e.g., CART cells,e.g., a mixture of cells expressing different CARs, in combination withanother agent, e.g., a kinase inhibitor, such as a kinase inhibitordescribed herein. In another aspect, the present invention providesmethods comprising administering a population of cells wherein at leastone cell in the population expresses a CAR having ananti-mesoothelinbinding domain as described herein, and a second cellexpressing another agent, e.g., an agent which enhances the activity orfitness of a CAR-expressing cell, in combination with another agent,e.g., a kinase inhibitor, such as a kinase inhibitor described herein.

Regulatable Chimeric Antigen Receptors

In some embodiments, a regulatable CAR (RCAR) where the CAR activity canbe controlled is desirable to optimize the safety and efficacy of a CARtherapy. There are many ways CAR activities can be regulated. Forexample, inducible apoptosis using, e.g., a caspase fused to adimerization domain (see, e.g., Di et al., N Egnl. J. Med. 2011 Nov. 3;365(18):1673-1683), can be used as a safety switch in the CAR therapy ofthe instant invention. In an aspect, a RCAR comprises a set ofpolypeptides, typically two in the simplest embodiments, in which thecomponents of a standard CAR described herein, e.g., an antigen bindingdomain and an intracellular signaling domain, are partitioned onseparate polypeptides or members. In some embodiments, the set ofpolypeptides include a dimerization switch that, upon the presence of adimerization molecule, can couple the polypeptides to one another, e.g.,can couple an antigen binding domain to an intracellular signalingdomain.

In an aspect, an RCAR comprises two polypeptides or members: 1) anintracellular signaling member comprising an intracellular signalingdomain, e.g., a primary intracellular signaling domain described herein,and a first switch domain; 2) an antigen binding member comprising anantigen binding domain, e.g., that targets mesothelin as describedherein, and a second switch domain. Optionally, the RCAR comprises atransmembrane domain described herein. In an embodiment, a transmembranedomain can be disposed on the intracellular signaling member, on theantigen binding member, or on both. (Unless otherwise indicated, whenmembers or elements of an RCAR are described herein, the order can be asprovided, but other orders are included as well. In other words, in anembodiment, the order is as set out in the text, but in otherembodiments, the order can be different. E.g., the order of elements onone side of a transmembrane region can be different from the example,e.g., the placement of a switch domain relative to a intracellularsignaling domain can be different, e.g., reversed).

In an embodiment, the first and second switch domains can form anintracellular or an extracellular dimerization switch. In an embodiment,the dimerization switch can be a homodimerization switch, e.g., wherethe first and second switch domain are the same, or a heterodimerizationswitch, e.g., where the first and second switch domain are differentfrom one another.

In embodiments, an RCAR can comprise a “multi switch.” A multi switchcan comprise heterodimerization switch domains or homodimerizationswitch domains. A multi switch comprises a plurality of, e.g., 2, 3, 4,5, 6, 7, 8, 9, or 10, switch domains, independently, on a first member,e.g., an antigen binding member, and a second member, e.g., anintracellular signaling member. In an embodiment, the first member cancomprise a plurality of first switch domains, e.g., FKBP-based switchdomains, and the second member can comprise a plurality of second switchdomains, e.g., FRB-based switch domains. In an embodiment, the firstmember can comprise a first and a second switch domain, e.g., aFKBP-based switch domain and a FRB-based switch domain, and the secondmember can comprise a first and a second switch domain, e.g., aFKBP-based switch domain and a FRB-based switch domain.

In an embodiment, the intracellular signaling member comprises one ormore intracellular signaling domains, e.g., a primary intracellularsignaling domain and one or more costimulatory signaling domains.

In an embodiment, the antigen binding member may comprise one or moreintracellular signaling domains, e.g., one or more costimulatorysignaling domains. In an embodiment, the antigen binding membercomprises a plurality, e.g., 2 or 3 costimulatory signaling domainsdescribed herein, e.g., selected from 41BB, CD28, CD27, ICOS, and OX40,and in embodiments, no primary intracellular signaling domain. In anembodiment, the antigen binding member comprises the followingcostimulatory signaling domains, from the extracellular to intracellulardirection: 41BB-CD27; 41BB-CD27; CD27-41BB; 41BB-CD28; CD28-41BB;OX40-CD28; CD28-OX40; CD28-41BB; or 41BB-CD28. In such embodiments, theintracellular binding member comprises a CD3zeta domain. In one suchembodiment the RCAR comprises (1) an antigen binding member comprising,an antigen binding domain, e.g., described herein, a transmembranedomain, and two costimulatory domains and a first switch domain; and (2)an intracellular signaling domain comprising a transmembrane domain ormembrane tethering domain and at least one primary intracellularsignaling domain, and a second switch domain.

An embodiment provides RCARs wherein the antigen binding member is nottethered to the surface of the CAR cell. This allows a cell having anintracellular signaling member to be conveniently paired with one ormore antigen binding domains, without transforming the cell with asequence that encodes the antigen binding member. In such embodiments,the RCAR comprises: 1) an intracellular signaling member comprising: afirst switch domain, a transmembrane domain, an intracellular signalingdomain, e.g., a primary intracellular signaling domain, and a firstswitch domain; and 2) an antigen binding member comprising: an antigenbinding domain, e.g., described herein, and a second switch domain,wherein the antigen binding member does not comprise a transmembranedomain or membrane tethering domain, and, optionally, does not comprisean intracellular signaling domain. In some embodiments, the RCAR mayfurther comprise 3) a second antigen binding member comprising: a secondantigen binding domain, e.g., a second antigen binding domain that bindsa different antigen than is bound by the antigen binding domain; and asecond switch domain.

Also provided herein are RCARs wherein the antigen binding membercomprises bispecific activation and targeting capacity. In thisembodiment, the antigen binding member can comprise a plurality, e.g.,2, 3, 4, or 5 antigen binding domains, e.g., scFvs, wherein each antigenbinding domain binds to a target antigen, e.g. different antigens or thesame antigen, e.g., the same or different epitopes on the same antigen.In an embodiment, the plurality of antigen binding domains are intandem, and optionally, a linker or hinge region is disposed betweeneach of the antigen binding domains. Suitable linkers and hinge regionsare described herein.

An embodiment provides RCARs having a configuration that allowsswitching of proliferation. In this embodiment, the RCAR comprises: 1)an intracellular signaling member comprising: optionally, atransmembrane domain or membrane tethering domain; one or moreco-stimulatory signaling domain, e.g., selected from 41BB, CD28, CD27,ICOS, and OX40, and a switch domain; and 2) an antigen binding membercomprising: an antigen binding domain, e.g., described herein, atransmembrane domain, and a primary intracellular signaling domain,e.g., a CD3zeta domain, wherein the antigen binding member does notcomprise a switch domain, or does not comprise a switch domain thatdimerizes with a switch domain on the intracellular signaling member. Inan embodiment, the antigen binding member does not comprise aco-stimulatory signaling domain. In an embodiment, the intracellularsignaling member comprises a switch domain from a homodimerizationswitch. In an embodiment, the intracellular signaling member comprises afirst switch domain of a heterodimerization switch and the RCARcomprises a second intracellular signaling member which comprises asecond switch domain of the heterodimerization switch. In suchembodiments, the second intracellular signaling member comprises thesame intracellular signaling domains as the intracellular signalingmember. In an embodiment, the dimerization switch is intracellular. Inan embodiment, the dimerization switch is extracellular.

In any of the RCAR configurations described here, the first and secondswitch domains comprise a FKBP/FRB-based switch as described herein.

Also provided herein are cells comprising an RCAR described herein. Anycell that is engineered to express a RCAR can be used as a RCARX cell.In an embodiment the RCARX cell is a T cell, and is referred to as aRCART cell. In an embodiment the RCARX cell is an NK cell, and isreferred to as a RCARN cell.

Also provided herein are nucleic acids and vectors comprising RCARencoding sequences. Sequence encoding various elements of an RCAR can bedisposed on the same nucleic acid molecule, e.g., the same plasmid orvector, e.g., viral vector, e.g., lentiviral vector. In an embodiment,(i) sequence encoding an antigen binding member and (ii) sequenceencoding an intracellular signaling member, can be present on the samenucleic acid, e.g., vector. Production of the corresponding proteins canbe achieved, e.g., by the use of separate promoters, or by the use of abicistronic transcription product (which can result in the production oftwo proteins by cleavage of a single translation product or by thetranslation of two separate protein products). In an embodiment, asequence encoding a cleavable peptide, e.g., a P2A or F2A sequence, isdisposed between (i) and (ii). In an embodiment, a sequence encoding anIRES, e.g., an EMCV or EV71 IRES, is disposed between (i) and (ii). Inthese embodiments, (i) and (ii) are transcribed as a single RNA. In anembodiment, a first promoter is operably linked to (i) and a secondpromoter is operably linked to (ii), such that (i) and (ii) aretranscribed as separate mRNAs.

Alternatively, the sequence encoding various elements of an RCAR can bedisposed on the different nucleic acid molecules, e.g., differentplasmids or vectors, e.g., viral vector, e.g., lentiviral vector. E.g.,the (i) sequence encoding an antigen binding member can be present on afirst nucleic acid, e.g., a first vector, and the (ii) sequence encodingan intracellular signaling member can be present on the second nucleicacid, e.g., the second vector.

Dimerization Switches

Dimerization switches can be non-covalent or covalent. In a non-covalentdimerization switch, the dimerization molecule promotes a non-covalentinteraction between the switch domains. In a covalent dimerizationswitch, the dimerization molecule promotes a covalent interactionbetween the switch domains.

In an embodiment, the RCAR comprises a FKBP/FRAP, or FKBP/FRB-baseddimerization switch. FKBP12 (FKBP, or FK506 binding protein) is anabundant cytoplasmic protein that serves as the initial intracellulartarget for the natural product immunosuppressive drug, rapamycin.Rapamycin binds to FKBP and to the large PI3K homolog FRAP (RAFT, mTOR).FRB is a 93 amino acid portion of FRAP, that is sufficient for bindingthe FKBP-rapamycin complex (Chen, J., Zheng, X. F., Brown, E. J. &Schreiber, S. L. (1995) Identification of an 11-kDaFKBP12-rapamycin-binding domain within the 289-kDa FKBP12-rapamycin-associated protein and characterization of a criticalserine residue. Proc Natl Acad Sci USA 92: 4947-51.)

In embodiments, an FKBP/FRAP, e.g., an FKBP/FRB, based switch can use adimerization molecule, e.g., rapamycin or a rapamycin analog.

The amino acid sequence of FKBP is as follows:

(SEQ ID NO: 382) D V P D Y A S L G G P S S P K K K R K V S R G V QV E T I S P G D G R T F P K R G Q T C V V H Y T GM L E D G K K F D S S R D R N K P F K F M L G K QE V I R G W E E G V A Q M S V G Q R A K L T I S PD Y A Y G A T G H P G I I P P H A T L V F D V E L L K L E T S Y

In embodiments, an FKBP switch domain can comprise a FRB bindingfragment of FKBP, e.g., the underlined portion of SEQ ID NO: 382, whichis:

(SEQ ID NO: 383) V Q V E T I S P G D G R T F P K R G Q T C V V H YT G M L E D G K K F D S S R D R N K P F K F M L GK Q E V I R G W E E G V A Q M S V G Q R A K L T IS P D Y A Y G A T G H P G I I P P H A T L V F D V E L L K L E T S

The amino acid sequence of FRB is as follows:

(SEQ ID NO: 384) ILWHEMWHEG LEEASRLYFG ERNVKGMFEV LEPLHAMMERGPQTLKETSF NQAYGRDLME AQEWCRKYMK SGNVKDLTQA WDLYYHVFRR ISK

“FKBP/FRAP, e.g., an FKPP/FRB, based switch” as that term is usedherein, refers to a dimerization switch comprising: a first switchdomain, comprises an FRB binding fragment or an FKBP analog, e.g.,RAD001, and has at least 70, 75, 80, 85, 90, 95, 96, 97, 98, or 99%identity with, or differs by no more than 30, 25, 20, 15, 10, 5, 4, 3,2, or 1 amino acid residues from, the FKBP sequence of SEQ ID NO: 382 or383; and a second switch domain, which comprises an FKBP bindingfragment or an FRB analog, and has at least 70, 75, 80, 85, 90, 95, 96,97, 98, or 99% identity with, or differs by no more than 30, 25, 20, 15,10, 5, 4, 3, 2, or 1 amino acid residues from, the FRB sequence of SEQID NO: 384. In an embodiment, a RCAR described herein comprises oneswitch domain comprises amino acid residues disclosed in SEQ ID NO: 382(or SEQ ID NO:383), and one switch domain comprises amino acid residuesdisclosed in SEQ ID NO: 384.

In embodiments, the FKBP/FRB dimerization switch comprises a modifiedFRB switch domain that exhibits altered, e.g., increased, affinity forthe dimerization molecule, e.g., rapamycin or a rapalogue, e.g., RAD001.In an embodiment, the modified FRB switch domain comprises one or moremutations, e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10 or more, selected frommutations at amino acid position(s)L2031, E2032, 52035, R2036, F2039,G2040, T2098, W2101, D2102, Y2105, and F2108, where the wild-type aminoacid is mutated to any other naturally-occurring amino acid. In anembodiment, a mutant FRB comprises a mutation at E2032, where E2032 ismutated to phenylalanine (E2032F), methionine (E2032M), arginine(E2032R), valine (E2032V), tyrosine (E2032Y), isoleucine (E20321), e.g.,SEQ ID NO: 385, or leucine (E2032L), e.g., SEQ ID NO: 386. In anembodiment, a mutant FRB comprises a mutation at T2098, where T2098 ismutated to phenylalanine (T2098F) or leucine (T2098L), e.g., SEQ IDNO:387. In an embodiment, a mutant FRB comprises a mutation at E2032 andat T2098, where E2032 is mutated to any amino acid, and where T2098 ismutated to any amino acid, e.g., SEQ ID NO: 388. In an embodiment, amutant FRB comprises an E20321 and a T2098L mutation, e.g., SEQ ID NO:389. In adn embodiment, a mutant FRB comprises an E2032L and a T2098Lmutation, e.g., SEQ ID NO: 340.

TABLE 15Exemplary mutant FRB having increased affinity for a dimerizationmolecule. SEQ ID FRB mutant Amino Acid Sequence NO: E2032I mutantILWHEMWHEGLIEASRLYFGERNVKGMFEVLEPLHAMMERGPQTLKETSFNQAYGR 385DLMEAQEWCRKYMKSGNVKDLTQAWDLYYHVFRRISKTS E2032L mutantILWHEMWHEGLLEASRLYFGERNVKGMFEVLEPLHAMMERGPQTLKETSFNQAYGR 386DLMEAQEWCRKYMKSGNVKDLTQAWDLYYHVFRRISKTS T2098L mutantILWHEMWHEGLEEASRLYFGERNVKGMFEVLEPLHAMMERGPQTLKETSFNQAYGR 387DLMEAQEWCRKYMKSGNVKDLLQAWDLYYHVFRRISKTS E2032, T2098 ILWHEMWHEGL XEASRLYFGERNVKGMFEVLEPLHAMMERGPQTLKETSFNQAYGR 388 mutantDLMEAQEWCRKYMKSGNVKDL X QAWDLYYHVFRRISKTS E20321, T2098LILWHEMWHEGLIEASRLYFGERNVKGMFEVLEPLHAMMERGPQTLKETSFNQAYGR 389 mutantDLMEAQEWCRKYMKSGNVKDLLQAWDLYYHVFRRISKTS E2032L, T2098LILWHEMWHEGLLEASRLYFGERNVKGMFEVLEPLHAMMERGPQTLKETSFNQAYGR 390 mutantDLMEAQEWCRKYMKSGNVKDLLQAWDLYYHVFRRISKTS

Other suitable dimerization switches include a GyrB-GyrB baseddimerization switch, a Gibberellin-based dimerization switch, atag/binder dimerization switch, and a halo-tag/snap-tag dimerizationswitch. Following the guidance provided herein, such switches andrelevant dimerization molecules will be apparent to one of ordinaryskill.

Dimerization Molecule

Association between the switch domains is promoted by the dimerizationmolecule. In the presence of dimerization molecule interaction orassociation between switch domains allows for signal transductionbetween a polypeptide associated with, e.g., fused to, a first switchdomain, and a polypeptide associated with, e.g., fused to, a secondswitch domain. In the presence of non-limiting levels of dimerizationmolecule signal transduction is increased by 1.1, 1.2, 1.3, 1.4, 1.5,1.6, 1.7, 1.8, 1.9, 2, 5, 10, 50, 100 fold, e.g., as measured in asystem described herein.

Rapamycin and rapamycin analogs (sometimes referred to as rapalogues),e.g., RAD001, can be used as dimerization molecules in a FKBP/FRB-baseddimerization switch described herein. In an embodiment the dimerizationmolecule can be selected from rapamycin (sirolimus), RAD001(everolimus), zotarolimus, temsirolimus, AP-23573 (ridaforolimus),biolimus and AP21967. Additional rapamycin analogs suitable for use withFKBP/FRB-based dimerization switches are further described in thesection entitled “Combination Therapies”, or in the subsection entitled“Exemplary mTOR inhibitors”.

RNA Transfection

Disclosed herein are methods for producing an in vitro transcribed RNACAR. The present invention also includes a CAR encoding RNA constructthat can be directly transfected into a cell. A method for generatingmRNA for use in transfection can involve in vitro transcription (IVT) ofa template with specially designed primers, followed by polyA addition,to produce a construct containing 3′ and 5′ untranslated sequence(“UTR”), a 5′ cap and/or Internal Ribosome Entry Site (IRES), thenucleic acid to be expressed, and a polyA tail, typically 50-2000 basesin length (SEQ ID NO:35). RNA so produced can efficiently transfectdifferent kinds of cells. In one aspect, the template includes sequencesfor the CAR.

In one aspect the mesothelin CAR is encoded by a messenger RNA (mRNA).In one aspect the mRNA encoding the mesothelin CAR is introduced into aT cell for production of a CART cell.

In one embodiment, the in vitro transcribed RNA CAR can be introduced toa cell as a form of transient transfection. The RNA is produced by invitro transcription using a polymerase chain reaction (PCR)-generatedtemplate. DNA of interest from any source can be directly converted byPCR into a template for in vitro mRNA synthesis using appropriateprimers and RNA polymerase. The source of the DNA can be, for example,genomic DNA, plasmid DNA, phage DNA, cDNA, synthetic DNA sequence or anyother appropriate source of DNA. The desired temple for in vitrotranscription is a CAR of the present invention. For example, thetemplate for the RNA CAR comprises an extracellular region comprising asingle chain variable domain of an anti-tumor antibody; a hinge region,a transmembrane domain (e.g., a transmembrane domain of CD8a); and acytoplasmic region that includes an intracellular signaling domain,e.g., comprising the signaling domain of CD3-zeta and the signalingdomain of 4-1BB.

In one embodiment, the DNA to be used for PCR contains an open readingframe. The DNA can be from a naturally occurring DNA sequence from thegenome of an organism. In one embodiment, the nucleic acid can includesome or all of the 5′ and/or 3′ untranslated regions (UTRs). The nucleicacid can include exons and introns. In one embodiment, the DNA to beused for PCR is a human nucleic acid sequence. In another embodiment,the DNA to be used for PCR is a human nucleic acid sequence includingthe 5′ and 3′ UTRs. The DNA can alternatively be an artificial DNAsequence that is not normally expressed in a naturally occurringorganism. An exemplary artificial DNA sequence is one that containsportions of genes that are ligated together to form an open readingframe that encodes a fusion protein. The portions of DNA that areligated together can be from a single organism or from more than oneorganism.

PCR is used to generate a template for in vitro transcription of mRNAwhich is used for transfection. Methods for performing PCR are wellknown in the art. Primers for use in PCR are designed to have regionsthat are substantially complementary to regions of the DNA to be used asa template for the PCR. The term “substantially complementary” refers tosequences of nucleotides where a majority or all of the bases in theprimer sequence are complementary, or one or more bases arenon-complementary, or mismatched. Substantially complementary sequencesare able to anneal or hybridize with the intended DNA target underannealing conditions used for PCR. The primers can be designed to besubstantially complementary to any portion of the DNA template. Forexample, the primers can be designed to amplify the portion of a nucleicacid that is normally transcribed in cells (the open reading frame),including 5′ and 3′ UTRs. The primers can also be designed to amplify aportion of a nucleic acid that encodes a particular domain of interest.In one embodiment, the primers are designed to amplify the coding regionof a human cDNA, including all or portions of the 5′ and 3′ UTRs.Primers useful for PCR can be generated by synthetic methods that arewell known in the art. “Forward primers” are primers that contain aregion of nucleotides that are substantially complementary tonucleotides on the DNA template that are upstream of the DNA sequencethat is to be amplified. The term “upstream” refers to a location 5′ tothe DNA sequence to be amplified relative to the coding strand. “Reverseprimers” are primers that contain a region of nucleotides that aresubstantially complementary to a double-stranded DNA template that aredownstream of the DNA sequence that is to be amplified. The term“downstream” refers to a location 3′ to the DNA sequence to be amplifiedrelative to the coding strand.

Any DNA polymerase useful for PCR can be used in the methods disclosedherein. The reagents and polymerase are commercially available from anumber of sources.

Chemical structures with the ability to promote stability and/ortranslation efficiency may also be used. The RNA preferably has 5′ and3′ UTRs. In one embodiment, the 5′ UTR is between one and 3000nucleotides in length. The length of 5′ and 3′ UTR sequences to be addedto the coding region can be altered by different methods, including, butnot limited to, designing primers for PCR that anneal to differentregions of the UTRs. Using this approach, one of ordinary skill in theart can modify the 5′ and 3′ UTR lengths required to achieve optimaltranslation efficiency following transfection of the transcribed RNA.

The 5′ and 3′ UTRs can be the naturally occurring, endogenous 5′ and 3′UTRs for the nucleic acid of interest. Alternatively, UTR sequences thatare not endogenous to the nucleic acid of interest can be added byincorporating the UTR sequences into the forward and reverse primers orby any other modifications of the template. The use of UTR sequencesthat are not endogenous to the nucleic acid of interest can be usefulfor modifying the stability and/or translation efficiency of the RNA.For example, it is known that AU-rich elements in 3′ UTR sequences candecrease the stability of mRNA. Therefore, 3′ UTRs can be selected ordesigned to increase the stability of the transcribed RNA based onproperties of UTRs that are well known in the art.

In one embodiment, the 5′ UTR can contain the Kozak sequence of theendogenous nucleic acid. Alternatively, when a 5′ UTR that is notendogenous to the nucleic acid of interest is being added by PCR asdescribed above, a consensus Kozak sequence can be redesigned by addingthe 5′ UTR sequence. Kozak sequences can increase the efficiency oftranslation of some RNA transcripts, but does not appear to be requiredfor all RNAs to enable efficient translation. The requirement for Kozaksequences for many mRNAs is known in the art. In other embodiments the5′ UTR can be 5′UTR of an RNA virus whose RNA genome is stable in cells.In other embodiments various nucleotide analogues can be used in the 3′or 5′ UTR to impede exonuclease degradation of the mRNA.

To enable synthesis of RNA from a DNA template without the need for genecloning, a promoter of transcription should be attached to the DNAtemplate upstream of the sequence to be transcribed. When a sequencethat functions as a promoter for an RNA polymerase is added to the 5′end of the forward primer, the RNA polymerase promoter becomesincorporated into the PCR product upstream of the open reading framethat is to be transcribed. In one preferred embodiment, the promoter isa T7 polymerase promoter, as described elsewhere herein. Other usefulpromoters include, but are not limited to, T3 and SP6 RNA polymerasepromoters. Consensus nucleotide sequences for T7, T3 and SP6 promotersare known in the art.

In a preferred embodiment, the mRNA has both a cap on the 5′ end and a3′ poly(A) tail which determine ribosome binding, initiation oftranslation and stability mRNA in the cell. On a circular DNA template,for instance, plasmid DNA, RNA polymerase produces a long concatamericproduct which is not suitable for expression in eukaryotic cells. Thetranscription of plasmid DNA linearized at the end of the 3′ UTR resultsin normal sized mRNA which is not effective in eukaryotic transfectioneven if it is polyadenylated after transcription.

On a linear DNA template, phage T7 RNA polymerase can extend the 3′ endof the transcript beyond the last base of the template (Schenborn andMierendorf, Nuc Acids Res., 13:6223-36 (1985); Nacheva andBerzal-Herranz, Eur. J. Biochem., 270:1485-65 (2003).

The conventional method of integration of polyA/T stretches into a DNAtemplate is molecular cloning. However polyA/T sequence integrated intoplasmid DNA can cause plasmid instability, which is why plasmid DNAtemplates obtained from bacterial cells are often highly contaminatedwith deletions and other aberrations. This makes cloning procedures notonly laborious and time consuming but often not reliable. That is why amethod which allows construction of DNA templates with polyA/T 3′stretch without cloning highly desirable.

The polyA/T segment of the transcriptional DNA template can be producedduring PCR by using a reverse primer containing a polyT tail, such as100T tail (SEQ ID NO: 31) (size can be 50-5000 T (SEQ ID NO: 32)), orafter PCR by any other method, including, but not limited to, DNAligation or in vitro recombination. Poly(A) tails also provide stabilityto RNAs and reduce their degradation. Generally, the length of a poly(A)tail positively correlates with the stability of the transcribed RNA. Inone embodiment, the poly(A) tail is between 100 and 5000 adenosines (SEQID NO: 33).

Poly(A) tails of RNAs can be further extended following in vitrotranscription with the use of a poly(A) polymerase, such as E. colipolyA polymerase (E-PAP). In one embodiment, increasing the length of apoly(A) tail from 100 nucleotides to between 300 and 400 nucleotides(SEQ ID NO: 34) results in about a two-fold increase in the translationefficiency of the RNA. Additionally, the attachment of differentchemical groups to the 3′ end can increase mRNA stability. Suchattachment can contain modified/artificial nucleotides, aptamers andother compounds. For example, ATP analogs can be incorporated into thepoly(A) tail using poly(A) polymerase. ATP analogs can further increasethe stability of the RNA.

5′ caps on also provide stability to RNA molecules. In a preferredembodiment, RNAs produced by the methods disclosed herein include a 5′cap. The 5′ cap is provided using techniques known in the art anddescribed herein (Cougot, et al., Trends in Biochem. Sci., 29:436-444(2001); Stepinski, et al., RNA, 7:1468-95 (2001); Elango, et al.,Biochim. Biophys. Res. Commun., 330:958-966 (2005)).

The RNAs produced by the methods disclosed herein can also contain aninternal ribosome entry site (IRES) sequence. The IRES sequence may beany viral, chromosomal or artificially designed sequence which initiatescap-independent ribosome binding to mRNA and facilitates the initiationof translation. Any solutes suitable for cell electroporation, which cancontain factors facilitating cellular permeability and viability such assugars, peptides, lipids, proteins, antioxidants, and surfactants can beincluded.

RNA can be introduced into target cells using any of a number ofdifferent methods, for instance, commercially available methods whichinclude, but are not limited to, electroporation (Amaxa Nucleofector-II(Amaxa Biosystems, Cologne, Germany)), (ECM 830 (BTX) (HarvardInstruments, Boston, Mass.) or the Gene Pulser II (BioRad, Denver,Colo.), Multiporator (Eppendort, Hamburg Germany), cationic liposomemediated transfection using lipofection, polymer encapsulation, peptidemediated transfection, or biolistic particle delivery systems such as“gene guns” (see, for example, Nishikawa, et al. Hum Gene Ther.,12(8):861-70 (2001).

Nucleic Acid Constructs Encoding a CAR

The present invention provides CAR transgenes comprising nucleic acidsequences encoding one or more CAR constructs of the invention. In oneaspect, the CAR transgene is provided as a messenger RNA transcript. Inone aspect, the CAR transgene is provided as a DNA construct.

Accordingly, in one aspect, the invention pertains to an isolatednucleic acid molecule encoding a chimeric antigen receptor (CAR),wherein the CAR comprises an anti-mesothelin binding domain (e.g., ahuman anti-mesothelin binding domain), a transmembrane domain, and anintracellular signaling domain comprising a stimulatory domain. In oneembodiment, the anti-mesothelin binding domain is an anti-mesothelinbinding domain described herein, e.g., an anti-mesothelin binding domainwhich comprises a sequence selected from a group consisting of SEQ IDNO: 87-111, or a sequence with 95-99% identify thereof. In oneembodiment, the isolated nucleic acid molecule further comprises asequence encoding a costimulatory domain. In one embodiment, thetransmembrane domain is a transmembrane domain of a protein selectedfrom the group consisting of the alpha, beta or zeta chain of the T-cellreceptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33,CD37, CD64, CD80, CD86, CD134, CD137 and CD154. In one embodiment, thetransmembrane domain comprises a sequence of SEQ ID NO: 6, or a sequencewith 95-99% identity thereof. In one embodiment, the anti-mesothelinbinding domain is connected to the transmembrane domain by a hingeregion, e.g., a hinge described herein. In one embodiment, the hingeregion comprises SEQ ID NO:2 or SEQ ID NO:3 or SEQ ID NO:4 or SEQ IDNO:5, or a sequence with 95-99% identity thereof. In one embodiment, theisolated nucleic acid molecule further comprises a sequence encoding acostimulatory domain. In one embodiment, the costimulatory domain is afunctional signaling domain of a protein selected from the groupconsisting of OX40, CD27, CD28, CD5, ICAM-1, LFA-1 (CD11a/CD18), ICOS(CD278), and 4-1BB (CD137). Further examples of such costimulatorymolecules include CDS, ICAM-1, GITR, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80(KLRF1), NKp44, NKp30, NKp46, CD160, CD19, CD4, CD8alpha, CD8beta, IL2Rbeta, IL2R gamma, IL7R alpha, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D,ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1,ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7,NKG2D, NKG2C, TNFR2, TRANCE/RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4),CD84, CD96 (Tactile), CEACAM1, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1,CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150,IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, LAT, GADS, SLP-76, andPAG/Cbp. In one embodiment, the costimulatory domain comprises asequence of SEQ ID NO:7, or a sequence with 95-99% identity thereof. Inone embodiment, the intracellular signaling domain comprises afunctional signaling domain of 4-1BB and a functional signaling domainof CD3 zeta. In one embodiment, the intracellular signaling domaincomprises the sequence of SEQ ID NO: 7 or SEQ ID NO: 8, or a sequencewith 95-99% identity thereof, and the sequence of SEQ ID NO: 9 or SEQ IDNO: 10, or a sequence with 95-99% identity thereof, wherein thesequences comprising the intracellular signaling domain are expressed inthe same frame and as a single polypeptide chain. In another aspect, theinvention pertains to an isolated nucleic acid molecule encoding a CARconstruct comprising a leader sequence of SEQ ID NO: 1, a scFv domainhaving a sequence selected from the group consisting of SEQ ID NO: 39;SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO:44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ IDNO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58,SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61, and SEQ ID NO: 62, (or asequence with 95-99% identify thereof), a hinge region of SEQ ID NO: 2or SEQ ID NO:3 or SEQ ID NO:4 or SEQ ID NO:5 (or a sequence with 95-99%identity thereof), a transmembrane domain having a sequence of SEQ IDNO: 6 (or a sequence with 95-99% identity thereof), a 4-1BBcostimulatory domain having a sequence of SEQ ID NO:7 (or a sequencewith 95-99% identity thereof) or a CD27 costimulatory domain having asequence of SEQ ID NO: 8 (or a sequence with 95-99% identity thereof),and a CD3 zeta stimulatory domain having a sequence of SEQ ID NO:9 orSEQ ID NO: 10 (or a sequence with 95-99% identity thereof).

In another aspect, the invention pertains to an isolated polypeptidemolecule encoded by the nucleic acid molecule. In one embodiment, theisolated polypeptide molecule comprises a sequence selected from thegroup consisting of SEQ ID NO: 63; SEQ ID NO: 64, SEQ ID NO: 65, SEQ IDNO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 70, SEQID NO: 71, SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 75,SEQ ID NO: 76, SEQ ID NO: 77, SEQ ID NO: 78, SEQ ID NO: 79, SEQ ID NO:80, SEQ ID NO: 81, SEQ ID NO: 82, SEQ ID NO: 83, SEQ ID NO: 84, SEQ IDNO: 85, and SEQ ID NO: 86, or a sequence with 95-99% identity thereof.

In another aspect, the invention pertains to an isolated nucleic acidmolecule encoding a chimeric antigen receptor (CAR) molecule thatcomprises an anti-mesothelin binding domain, a transmembrane domain, andan intracellular signaling domain comprising a stimulatory domain, andwherein the nucleic acid encoding the anti-mesothelin binding domaincomprises a sequence selected from the group consisting of SEQ ID NO:111; SEQ ID NO: 112, SEQ ID NO: 113, SEQ ID NO: 114, SEQ ID NO: 115, SEQID NO: 116, SEQ ID NO: 117, SEQ ID NO: 118, SEQ ID NO: 119, SEQ ID NO:120, SEQ ID NO: 121, SEQ ID NO: 122, SEQ ID NO: 123, SEQ ID NO: 124, SEQID NO: 125, SEQ ID NO: 126, SEQ ID NO: 127, SEQ ID NO: 128, SEQ ID NO:129, SEQ ID NO: 130, SEQ ID NO: 131, SEQ ID NO: 132, SEQ ID NO: 133, SEQID NO:134, or a sequence with 95-99% identify thereof.

In one embodiment, the encoded CAR molecule further comprises a sequenceencoding a costimulatory domain. In one embodiment, the costimulatorydomain is a functional signaling domain of a protein selected from thegroup consisting of OX40, CD27, CD28, CDS, ICAM-1, LFA-1 (CD11a/CD18)and 4-1BB (CD137). In one embodiment, the costimulatory domain comprisesa sequence of SEQ ID NO:7. In one embodiment, the transmembrane domainis a transmembrane domain of a protein selected from the groupconsisting of the alpha, beta or zeta chain of the T-cell receptor,CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37,CD64, CD80, CD86, CD134, CD137 and CD154. In one embodiment, thetransmembrane domain comprises a sequence of SEQ ID NO:6. In oneembodiment, the intracellular signaling domain comprises a functionalsignaling domain of 4-1BB and a functional signaling domain of zeta. Inone embodiment, the intracellular signaling domain comprises thesequence of SEQ ID NO: 7 and the sequence of SEQ ID NO: 9, wherein thesequences comprising the intracellular signaling domain are expressed inthe same frame and as a single polypeptide chain. In one embodiment, theanti-mesothelin binding domain is connected to the transmembrane domainby a hinge region. In one embodiment, the hinge region comprises SEQ IDNO:2. In one embodiment, the hinge region comprises SEQ ID NO: 3, SEQ IDNO: 4, or SEQ ID NO: 5.

In another aspect, the invention pertains to an isolated CAR moleculecomprising a leader sequence of SEQ ID NO: 1, a scFv domain having asequence selected from the group consisting of SEQ ID NOS: 39-62, or asequence with 95-99% identify thereof, a hinge region of SEQ ID NO:2 orSEQ ID NO: 3 or SEQ ID NO: 4 or SEQ ID NO: 5, a transmembrane domainhaving a sequence of SEQ ID NO: 6, a 4-1BB costimulatory domain having asequence of SEQ ID NO:7 or a CD27 costimulatory domain having a sequenceof SEQ ID NO: 8, and a CD3 zeta stimulatory domain having a sequence ofSEQ ID NO:9 or SEQ ID NO: 10. In one embodiment, the encoded CARmolecule comprises a sequence selected from the group consisting of SEQID NOS: 63-86, or a sequence with 95-99% identify thereof.

The present invention further provides vectors comprising CARtransgenes. In one aspect, a CAR vectors can be directly transduced intoa cell, e.g., a T cell or NK cell. In one aspect, the vector is acloning or expression vector, e.g., a vector including, but not limitedto, one or more plasmids (e.g., expression plasmids, cloning vectors,minicircles, minivectors, double minute chromosomes), retroviral andlentiviral vector constructs. In one aspect, the vector is capable ofexpressing the CAR construct in mammalian T cells or NK cells. In oneaspect, the mammalian T cell is a human T cell or a human NK cell.

The present invention also includes a CAR encoding RNA construct thatcan be directly transfected into a cell, e.g., a T cell or a NK cell. Amethod for generating mRNA for use in transfection involves in vitrotranscription (IVT) of a template with specially designed primers,followed by polyA addition, to produce a construct containing 3′ and 5′untranslated sequence (“UTR”), a 5′ cap and/or Internal Ribosome EntrySite (IRES), the gene to be expressed, and a polyA tail, typically50-2000 bases in length. RNA so produced can efficiently transfectdifferent kinds of cells. In one aspect, the template includes sequencesfor the CAR.

In one aspect the mesothelin CAR transgene is encoded by a messenger RNA(mRNA). In one aspect the mRNA encoding the mesothelin CAR transgene isintroduced into a T cell for production of a CART cell, or a NK cell.

Vectors

The present invention also provides vectors in which a DNA of thepresent invention is inserted. Vectors derived from retroviruses such asthe lentivirus are suitable tools to achieve long-term gene transfersince they allow long-term, stable integration of a transgene and itspropagation in daughter cells. Lentiviral vectors have the addedadvantage over vectors derived from onco-retroviruses such as murineleukemia viruses in that they can transduce non-proliferating cells,such as hepatocytes. They also have the added advantage of lowimmunogenicity.

In one embodiment, the vector comprising the nucleic acid encoding thedesired CAR of the invention is a DNA, a RNA, a plasmid, an adenoviralvector, a lentivirus vector, or a retrovirus vector.

In another embodiment, the vector comprising the nucleic acid encodingthe desired CAR of the invention is an adenoviral vector (A5/35). Inanother embodiment, the expression of nucleic acids encoding CARs can beaccomplished using of transposons such as sleeping beauty, CRISPR, CAS9,and zinc finger nucleases. See, e.g., June et al. 2009 Nature ReviewsImmunology 9.10: 704-716, incorporated herein by reference in itsentirety.

In brief summary, the expression of natural or synthetic nucleic acidsencoding CARs is typically achieved by operably linking a nucleic acidencoding the CAR polypeptide or portions thereof to a promoter, andincorporating the construct into an expression vector. The vectors canbe suitable for replication and integration eukaryotes. Typical cloningvectors contain transcription and translation terminators, initiationsequences, and promoters useful for regulation of the expression of thedesired nucleic acid sequence.

The expression constructs of the present invention may also be used fornucleic acid immunization and gene therapy, using standard gene deliveryprotocols. Methods for gene delivery are known in the art. See, e.g.,U.S. Pat. Nos. 5,399,346, 5,580,859, 5,589,466, incorporated byreference herein in their entireties. In another embodiment, theinvention provides a gene therapy vector.

The nucleic acid can be cloned into a number of types of vectors. Forexample, the nucleic acid can be cloned into a vector including, but notlimited to a plasmid, a phagemid, a phage derivative, an animal virus,and a cosmid. Vectors of particular interest include expression vectors,replication vectors, probe generation vectors, and sequencing vectors.

Further, the expression vector may be provided to a cell in the form ofa viral vector. Viral vector technology is well known in the art and isdescribed, for example, in Sambrook et al., 2012, MOLECULAR CLONING: ALABORATORY MANUAL, volumes 1-4, Cold Spring Harbor Press, NY), and inother virology and molecular biology manuals. Viruses, which are usefulas vectors include, but are not limited to, retroviruses, adenoviruses,adeno-associated viruses, herpes viruses, and lentiviruses. In general,a suitable vector contains an origin of replication functional in atleast one organism, a promoter sequence, convenient restrictionendonuclease sites, and one or more selectable markers, (e.g., WO01/96584; WO 01/29058; and U.S. Pat. No. 6,326,193).

A number of viral based systems have been developed for gene transferinto mammalian cells. For example, retroviruses provide a convenientplatform for gene delivery systems. A selected gene can be inserted intoa vector and packaged in retroviral particles using techniques known inthe art. The recombinant virus can then be isolated and delivered tocells of the subject either in vivo or ex vivo. A number of retroviralsystems are known in the art. In some embodiments, adenovirus vectorsare used. A number of adenovirus vectors are known in the art. In oneembodiment, lentivirus vectors are used.

Additional promoter elements, e.g., enhancers, regulate the frequency oftranscriptional initiation. Typically, these are located in the region30-110 bp upstream of the start site, although a number of promotershave been shown to contain functional elements downstream of the startsite as well. The spacing between promoter elements frequently isflexible, so that promoter function is preserved when elements areinverted or moved relative to one another. In the thymidine kinase (tk)promoter, the spacing between promoter elements can be increased to 50bp apart before activity begins to decline. Depending on the promoter,it appears that individual elements can function either cooperatively orindependently to activate transcription. Exemplary promoters include theCMV IE gene, EF-1α, ubiquitin C, or phosphoglycerokinase (PGK)promoters.

An example of a promoter that is capable of expressing a CAR transgenein a mammalian T cell is the EF1alpha promoter (EF1a or EF1α). Thenative EF1a promoter drives expression of the alpha subunit of theelongation factor-1 complex, which is responsible for the enzymaticdelivery of aminoacyl tRNAs to the ribosome. The EF1a promoter has beenextensively used in mammalian expression plasmids and has been shown tobe effective in driving CAR expression from transgenes cloned into alentiviral vector. See, e.g., Milone et al., Mol. Ther. 17(8): 1453-1464(2009). In one aspect, the EF1a promoter comprises the sequence providedas SEQ ID NO:11.

Another example of a promoter is the immediate early cytomegalovirus(CMV) promoter sequence. This promoter sequence is a strong constitutivepromoter sequence capable of driving high levels of expression of anypolynucleotide sequence operatively linked thereto. However, otherconstitutive promoter sequences may also be used, including, but notlimited to the simian virus 40 (SV40) early promoter, mouse mammarytumor virus (MMTV), human immunodeficiency virus (HIV) long terminalrepeat (LTR) promoter, MoMuLV promoter, an avian leukemia viruspromoter, an Epstein-Barr virus immediate early promoter, a Rous sarcomavirus promoter, as well as human gene promoters such as, but not limitedto, the actin promoter, the myosin promoter, the elongation factor-1apromoter, the hemoglobin promoter, and the creatine kinase promoter.Further, the invention should not be limited to the use of constitutivepromoters. Inducible promoters are also contemplated as part of theinvention. The use of an inducible promoter provides a molecular switchcapable of turning on expression of the polynucleotide sequence which itis operatively linked when such expression is desired, or turning offthe expression when expression is not desired. Examples of induciblepromoters include, but are not limited to a metallothionine promoter, aglucocorticoid promoter, a progesterone promoter, and a tetracyclinepromoter.

In order to assess the expression of a CAR polypeptide or portionsthereof, the expression vector to be introduced into a cell can alsocontain either a selectable marker gene or a reporter gene or both tofacilitate identification and selection of expressing cells from thepopulation of cells sought to be transfected or infected through viralvectors. In other aspects, the selectable marker may be carried on aseparate piece of DNA and used in a co-transfection procedure. Bothselectable markers and reporter genes may be flanked with appropriateregulatory sequences to enable expression in the host cells. Usefulselectable markers include, for example, antibiotic-resistance genes,such as neo and the like.

Reporter genes are used for identifying potentially transfected cellsand for evaluating the functionality of regulatory sequences. Ingeneral, a reporter gene is a gene that is not present in or expressedby the recipient organism or tissue and that encodes a polypeptide whoseexpression is manifested by some easily detectable property, e.g.,enzymatic activity. Expression of the reporter gene is assayed at asuitable time after the DNA has been introduced into the recipientcells. Suitable reporter genes may include genes encoding luciferase,beta-galactosidase, chloramphenicol acetyl transferase, secretedalkaline phosphatase, or the green fluorescent protein gene (e.g.,Ui-Tei et al., 2000 FEBS Letters 479: 79-82). Suitable expressionsystems are well known and may be prepared using known techniques orobtained commercially. In general, the construct with the minimal 5′flanking region showing the highest level of expression of reporter geneis identified as the promoter. Such promoter regions may be linked to areporter gene and used to evaluate agents for the ability to modulatepromoter-driven transcription.

In one embodiment, the vector can further comprise a nucleic acidencoding a second CAR. In one embodiment, the second CAR includes anantigen binding domain to, e.g., a target other than mesothelin onstroma cells, e.g., FAP; a target other than mesothelin on prostatecancer cells, e.g., androgen receptor, OR51E2, PSMA, PSCA, PDGRF-β,TARP, GloboH, MAD-CT-1, or MAD-CT-2; a target other than mesothelin onovararian cancer cells, e.g., Tn, PRSS21, CD171, Lewis Y, folatereceptor α, claudin6, GloboH, or sperm protein 17; e.g., a target otherthan mesothelin on lung cancer cells, e.g., VEGF, HER3, IGF-1R, EGFR,DLL4, or Trop-2. In one embodiment, the vector comprises a nucleic acidsequence encoding a first CAR that targets a first antigen and includesan intracellular signaling domain having a costimulatory signalingdomain but not a primary signaling domain, and a nucleic acid encoding asecond CAR that targets a second, different, antigen and includes anintracellular signaling domain having a primary signaling domain but nota costimulatory signaling domain. In one embodiment, the vectorcomprises a nucleic acid encoding a first mesothelin CAR that includes amesothelin binding domain, a transmembrane domain and a costimulatorydomain and a nucleic acid encoding a second CAR that targets an antigenother than mesothelin (e.g., a target other than mesothelin on stromacells, e.g., FAP; a target other than mesothelin on prostate cancercells, e.g., androgen receptor, OR51E2, PSMA, PSCA, PDGRF-β, TARP,GloboH, MAD-CT-1, or MAD-CT-2; a target other than mesothelin onovararian cancer cells, e.g., Tn, PRSS21, CD171, Lewis Y, folatereceptor α, claudin6, GloboH, or sperm protein 17; e.g., a target otherthan mesothelin on lung cancer cells, e.g., VEGF, HER3, IGF-1R, EGFR,DLL4, or Trop-2) and includes an antigen binding domain, a transmembranedomain and a primary signaling domain. In another embodiment, the vectorcomprises a nucleic acid encoding a first mesothelin CAR that includes amesothelin binding domain, a transmembrane domain and a primarysignaling domain and a nucleic acid encoding a second CAR that targetsan antigen other than mesothelin (e.g., a target other than mesothelinon stroma cells, e.g., FAP; a target other than mesothelin on prostatecancer cells, e.g., androgen receptor, OR51E2, PSMA, PSCA, PDGRF-β,TARP, GloboH, MAD-CT-1, or MAD-CT-2; a target other than mesothelin onovararian cancer cells, e.g., Tn, PRSS21, CD171, Lewis Y, folatereceptor α, claudin6, GloboH, or sperm protein 17; e.g., a target otherthan mesothelin on lung cancer cells, e.g., VEGF, HER3, IGF-1R, EGFR,DLL4, or Trop-2) and includes an antigen binding domain to the antigen,a transmembrane domain and a costimulatory signaling domain.

In one embodiment, the vector comprises a nucleic acid encoding amesothelin CAR described herein and a nucleic acid encoding aninhibitory CAR. In one embodiment, the inhibitory CAR comprises anantigen binding domain that binds an antigen found on normal cells butnot cancer cells, e.g., normal cells that also express CLL. In oneembodiment, the inhibitory CAR comprises the antigen binding domain, atransmembrane domain and an intracellular domain of an inhibitorymolecule. For example, the intracellular domain of the inhibitory CARcan be an intracellular domain of PD1, PD-L1, CTLA4, TIM3, CEACAM (e.g.,CEACAM-1, CEACAM-3 and/or CEACAM-5), LAG3, VISTA, BTLA, TIGIT, LAIR1,CD160, 2B4 and TGFR beta.

In one embodiment, the vector comprises a nucleic acid encoding amesothelin CAR described herein and an inhibitory nucleic acid, e.g., adsRNA, e.g., an siRNA or shRNA, e.g., as described herein.

Methods of introducing and expressing genes into a cell are known in theart. In the context of an expression vector, the vector can be readilyintroduced into a host cell, e.g., mammalian, bacterial, yeast, orinsect cell by any method in the art. For example, the expression vectorcan be transferred into a host cell by physical, chemical, or biologicalmeans.

Physical methods for introducing a polynucleotide into a host cellinclude calcium phosphate precipitation, lipofection, particlebombardment, microinjection, electroporation, and the like. Methods forproducing cells comprising vectors and/or exogenous nucleic acids arewell-known in the art. See, for example, Sambrook et al., 2012,MOLECULAR CLONING: A LABORATORY MANUAL, volumes 1-4, Cold Spring HarborPress, NY). A preferred method for the introduction of a polynucleotideinto a host cell is lipofection, e.g., using Lipofectamine (LifeTechnologies).

Biological methods for introducing a polynucleotide of interest into ahost cell include the use of DNA and RNA vectors. Viral vectors, andespecially retroviral vectors, have become the most widely used methodfor inserting genes into mammalian, e.g., human cells. Other viralvectors can be derived from lentivirus, poxviruses, herpes simplex virusI, adenoviruses and adeno-associated viruses, and the like. See, forexample, U.S. Pat. Nos. 5,350,674 and 5,585,362.

Chemical means for introducing a polynucleotide into a host cell includecolloidal dispersion systems, such as macromolecule complexes,nanocapsules, microspheres, beads, and lipid-based systems includingoil-in-water emulsions, micelles, mixed micelles, and liposomes. Anexemplary colloidal system for use as a delivery vehicle in vitro and invivo is a liposome (e.g., an artificial membrane vesicle). Other methodsof state-of-the-art targeted delivery of nucleic acids are available,such as delivery of polynucleotides with targeted nanoparticles or othersuitable sub-micron sized delivery system.

In the case where a non-viral delivery system is utilized, an exemplarydelivery vehicle is a liposome. The use of lipid formulations iscontemplated for the introduction of the nucleic acids into a host cell(in vitro, ex vivo or in vivo). In another aspect, the nucleic acid maybe associated with a lipid. The nucleic acid associated with a lipid maybe encapsulated in the aqueous interior of a liposome, interspersedwithin the lipid bilayer of a liposome, attached to a liposome via alinking molecule that is associated with both the liposome and theoligonucleotide, entrapped in a liposome, complexed with a liposome,dispersed in a solution containing a lipid, mixed with a lipid, combinedwith a lipid, contained as a suspension in a lipid, contained orcomplexed with a micelle, or otherwise associated with a lipid. Lipid,lipid/DNA or lipid/expression vector associated compositions are notlimited to any particular structure in solution. For example, they maybe present in a bilayer structure, as micelles, or with a “collapsed”structure. They may also simply be interspersed in a solution, possiblyforming aggregates that are not uniform in size or shape. Lipids arefatty substances which may be naturally occurring or synthetic lipids.For example, lipids include the fatty droplets that naturally occur inthe cytoplasm as well as the class of compounds which contain long-chainaliphatic hydrocarbons and their derivatives, such as fatty acids,alcohols, amines, amino alcohols, and aldehydes.

Lipids suitable for use can be obtained from commercial sources. Forexample, dimyristyl phosphatidylcholine (“DMPC”) can be obtained fromSigma, St. Louis, Mo.; dicetyl phosphate (“DCP”) can be obtained from K& K Laboratories (Plainview, N.Y.); cholesterol (“Choi”) can be obtainedfrom Calbiochem-Behring; dimyristyl phosphatidylglycerol (“DMPG”) andother lipids may be obtained from Avanti Polar Lipids, Inc. (Birmingham,Ala.). Stock solutions of lipids in chloroform or chloroform/methanolcan be stored at about −20° C. Chloroform is used as the only solventsince it is more readily evaporated than methanol. “Liposome” is ageneric term encompassing a variety of single and multilamellar lipidvehicles formed by the generation of enclosed lipid bilayers oraggregates. Liposomes can be characterized as having vesicularstructures with a phospholipid bilayer membrane and an inner aqueousmedium. Multilamellar liposomes have multiple lipid layers separated byaqueous medium. They form spontaneously when phospholipids are suspendedin an excess of aqueous solution. The lipid components undergoself-rearrangement before the formation of closed structures and entrapwater and dissolved solutes between the lipid bilayers (Ghosh et al.,1991 Glycobiology 5: 505-10). However, compositions that have differentstructures in solution than the normal vesicular structure are alsoencompassed. For example, the lipids may assume a micellar structure ormerely exist as nonuniform aggregates of lipid molecules. Alsocontemplated are lipofectamine-nucleic acid complexes.

Regardless of the method used to introduce exogenous nucleic acids intoa host cell or otherwise expose a cell to the inhibitor of the presentinvention, in order to confirm the presence of the recombinant DNAsequence in the host cell, a variety of assays may be performed. Suchassays include, for example, “molecular biological” assays well known tothose of skill in the art, such as Southern and Northern blotting,RT-PCR and PCR; “biochemical” assays, such as detecting the presence orabsence of a particular peptide, e.g., by immunological means (ELISAsand Western blots) or by assays described herein to identify agentsfalling within the scope of the invention.

The present invention further provides a vector comprising a CARencoding nucleic acid molecule. In one aspect, a CAR vector can bedirectly transduced into a cell, e.g., a T cell or a NK cell. In oneaspect, the vector is a cloning or expression vector, e.g., a vectorincluding, but not limited to, one or more plasmids (e.g., expressionplasmids, cloning vectors, minicircles, minivectors, double minutechromosomes), retroviral and lentiviral vector constructs. In oneaspect, the vector is capable of expressing the CAR construct inmammalian T cells. In one aspect, the mammalian T cell is a human Tcell. In one aspect, the mammalian cell is a human NK cell.

Sources of Cells

Prior to expansion and genetic modification, a source of cells (e.g., Tcells or NK cells) is obtained from a subject. The term “subject” isintended to include living organisms in which an immune response can beelicited (e.g., mammals). Examples of subjects include humans, dogs,cats, mice, rats, and transgenic species thereof. T cells can beobtained from a number of sources, including peripheral bloodmononuclear cells, bone marrow, lymph node tissue, cord blood, thymustissue, tissue from a site of infection, ascites, pleural effusion,spleen tissue, and tumors. In certain aspects of the present invention,any number of T cell lines available in the art, may be used. In certainaspects of the present invention, T cells can be obtained from a unit ofblood collected from a subject using any number of techniques known tothe skilled artisan, such as Ficoll™ separation. In one preferredaspect, cells from the circulating blood of an individual are obtainedby apheresis. The apheresis product typically contains lymphocytes,including T cells, monocytes, granulocytes, B cells, other nucleatedwhite blood cells, red blood cells, and platelets. In one aspect, thecells collected by apheresis may be washed to remove the plasma fractionand to place the cells in an appropriate buffer or media for subsequentprocessing steps. In one aspect of the invention, the cells are washedwith phosphate buffered saline (PBS). In an alternative aspect, the washsolution lacks calcium and may lack magnesium or may lack many if notall divalent cations. Initial activation steps in the absence of calciumcan lead to magnified activation. As those of ordinary skill in the artwould readily appreciate a washing step may be accomplished by methodsknown to those in the art, such as by using a semi-automated“flow-through” centrifuge (for example, the Cobe 2991 cell processor,the Baxter CytoMate, or the Haemonetics Cell Saver 5) according to themanufacturer's instructions. After washing, the cells may be resuspendedin a variety of biocompatible buffers, such as, for example, Ca-free,Mg-free PBS, PlasmaLyte A, or other saline solution with or withoutbuffer. Alternatively, the undesirable components of the apheresissample may be removed and the cells directly resuspended in culturemedia.

In one aspect, T cells are isolated from peripheral blood lymphocytes bylysing the red blood cells and depleting the monocytes, for example, bycentrifugation through a PERCOLL™ gradient or by counterflow centrifugalelutriation. A specific subpopulation of T cells, such as CD3+, CD28+,CD4+, CD8+, CD45RA+, and CD45RO+ T cells, can be further isolated bypositive or negative selection techniques. For example, in one aspect, Tcells are isolated by incubation with anti-CD3/anti-CD28 (e.g.,3×28)-conjugated beads, such as DYNABEADS® M-450 CD3/CD28 T, for a timeperiod sufficient for positive selection of the desired T cells. In oneaspect, the time period is about 30 minutes. In a further aspect, thetime period ranges from 30 minutes to 36 hours or longer and all integervalues there between. In a further aspect, the time period is at least1, 2, 3, 4, 5, or 6 hours. In yet another preferred aspect, the timeperiod is 10 to 24 hours. In one aspect, the incubation time period is24 hours. For isolation of T cells from patients with leukemia, use oflonger incubation times, such as 24 hours, can increase cell yield.Longer incubation times may be used to isolate T cells in any situationwhere there are few T cells as compared to other cell types, such inisolating tumor infiltrating lymphocytes (TIL) from tumor tissue or fromimmunocompromised individuals. Further, use of longer incubation timescan increase the efficiency of capture of CD8+ T cells. Thus, by simplyshortening or lengthening the time T cells are allowed to bind to theCD3/CD28 beads and/or by increasing or decreasing the ratio of beads toT cells (as described further herein), subpopulations of T cells can bepreferentially selected for or against at culture initiation or at othertime points during the process. Additionally, by increasing ordecreasing the ratio of anti-CD3 and/or anti-CD28 antibodies on thebeads or other surface, subpopulations of T cells can be preferentiallyselected for or against at culture initiation or at other desired timepoints. The skilled artisan would recognize that multiple rounds ofselection can also be used in the context of this invention. In certainaspects, it may be desirable to perform the selection procedure and usethe “unselected” cells in the activation and expansion process.“Unselected” cells can also be subjected to further rounds of selection.

Enrichment of a T cell population by negative selection can beaccomplished with a combination of antibodies directed to surfacemarkers unique to the negatively selected cells. One method is cellsorting and/or selection via negative magnetic immunoadherence or flowcytometry that uses a cocktail of monoclonal antibodies directed to cellsurface markers present on the cells negatively selected. For example,to enrich for CD4+ cells by negative selection, a monoclonal antibodycocktail typically includes antibodies to CD14, CD20, CD11b, CD16,HLA-DR, and CD8. In certain aspects, it may be desirable to enrich foror positively select for regulatory T cells which typically expressCD4+, CD25+, CD62Lhi, GITR+, and FoxP3+. Alternatively, in certainaspects, T regulatory cells are depleted by anti-C25 conjugated beads orother similar method of selection.

In one embodiment, a T cell population can be selected that expressesone or more of IFN-γ, TNFα, IL-17A, IL-2, IL-3, IL-4, GM-CSF, IL-10,IL-13, granzyme B, and perforin, or other appropriate molecules, e.g.,other cytokines. Methods for screening for cell expression can bedetermined, e.g., by the methods described in PCT Publication No.: WO2013/126712.

In one embodiment, a T cell population is diaglycerol kinase(DGK)-deficient. DGK-deficient cells include cells that do not expressDGK RNA or protein, or have reduced or inhibited DGK activity.DGK-deficient cells can be generated by genetic approaches, e.g.,administering RNA-interfering agents, e.g., siRNA, shRNA, miRNA, toreduce or prevent DGK expression. Alternatively, DGK-deficient cells canbe generated by treatment with DGK inhibitors described herein.

In one embodiment, a T cell population is Ikaros-deficient.Ikaros-deficient cells include cells that do not express Ikaros RNA orprotein, or have reduced or inhibited Ikaros activity, Ikaros-deficientcells can be generated by genetic approaches, e.g., administeringRNA-interfering agents, e.g., siRNA, shRNA, miRNA, to reduce or preventIkaros expression. Alternatively, Ikaros-deficient cells can begenerated by treatment with Ikaros inhibitors, e.g., lenalidomide.

In embodiments, a T cell population is DGK-deficient andIkaros-deficient, e.g., does not express DGK and Ikaros, or has reducedor inhibited DGK and Ikaros activity. Such DGK and Ikaros-deficientcells can be generated by any of the methods described herein.

For isolation of a desired population of cells by positive or negativeselection, the concentration of cells and surface (e.g., particles suchas beads) can be varied. In certain aspects, it may be desirable tosignificantly decrease the volume in which beads and cells are mixedtogether (e.g., increase the concentration of cells), to ensure maximumcontact of cells and beads. For example, in one aspect, a concentrationof 2 billion cells/ml is used. In one aspect, a concentration of 1billion cells/ml is used. In a further aspect, greater than 100 millioncells/ml is used. In a further aspect, a concentration of cells of 10,15, 20, 25, 30, 35, 40, 45, or 50 million cells/ml is used. In yet oneaspect, a concentration of cells from 75, 80, 85, 90, 95, or 100 millioncells/ml is used. In further aspects, concentrations of 125 or 150million cells/ml can be used. Using high concentrations can result inincreased cell yield, cell activation, and cell expansion. Further, useof high cell concentrations allows more efficient capture of cells thatmay weakly express target antigens of interest, such as CD28-negative Tcells, or from samples where there are many tumor cells present (e.g.,leukemic blood, tumor tissue, etc.). Such populations of cells may havetherapeutic value and would be desirable to obtain. For example, usinghigh concentration of cells allows more efficient selection of CD8+ Tcells that normally have weaker CD28 expression.

In a related aspect, it may be desirable to use lower concentrations ofcells. By significantly diluting the mixture of T cells and surface(e.g., particles such as beads), interactions between the particles andcells is minimized. This selects for cells that express high amounts ofdesired antigens to be bound to the particles. For example, CD4+ T cellsexpress higher levels of CD28 and are more efficiently captured thanCD8+ T cells in dilute concentrations. In one aspect, the concentrationof cells used is 5×10e⁶/ml. In other aspects, the concentration used canbe from about 1×10⁵/ml to 1×10⁶/ml, and any integer value in between.

In other aspects, the cells may be incubated on a rotator for varyinglengths of time at varying speeds at either 2-10° C. or at roomtemperature.

T cells for stimulation can also be frozen after a washing step. Wishingnot to be bound by theory, the freeze and subsequent thaw step providesa more uniform product by removing granulocytes and to some extentmonocytes in the cell population. After the washing step that removesplasma and platelets, the cells may be suspended in a freezing solution.While many freezing solutions and parameters are known in the art andwill be useful in this context, one method involves using PBS containing20% DMSO and 8% human serum albumin, or culture media containing 10%Dextran 40 and 5% Dextrose, 20% Human Serum Albumin and 7.5% DMSO, or31.25% Plasmalyte-A, 31.25% Dextrose 5%, 0.45% NaCl, 10% Dextran 40 and5% Dextrose, 20% Human Serum Albumin, and 7.5% DMSO or other suitablecell freezing media containing for example, Hespan and PlasmaLyte A, thecells then are frozen to −80° C. at a rate of 1° per minute and storedin the vapor phase of a liquid nitrogen storage tank. Other methods ofcontrolled freezing may be used as well as uncontrolled freezingimmediately at −20° C. or in liquid nitrogen.

In certain aspects, cryopreserved cells are thawed and washed asdescribed herein and allowed to rest for one hour at room temperatureprior to activation using the methods of the present invention.

Also contemplated in the context of the invention is the collection ofblood samples or apheresis product from a subject at a time period priorto when the expanded cells as described herein might be needed. As such,the source of the cells to be expanded can be collected at any timepoint necessary, and desired cells, such as T cells, isolated and frozenfor later use in T cell therapy for any number of diseases or conditionsthat would benefit from T cell therapy, such as those described herein.In one aspect a blood sample or an apheresis is taken from a generallyhealthy subject. In certain aspects, a blood sample or an apheresis istaken from a generally healthy subject who is at risk of developing adisease, but who has not yet developed a disease, and the cells ofinterest are isolated and frozen for later use. In certain aspects, theT cells may be expanded, frozen, and used at a later time. In certainaspects, samples are collected from a patient shortly after diagnosis ofa particular disease as described herein but prior to any treatments. Ina further aspect, the cells are isolated from a blood sample or anapheresis from a subject prior to any number of relevant treatmentmodalities, including but not limited to treatment with agents such asnatalizumab, efalizumab, antiviral agents, chemotherapy, radiation,immunosuppressive agents, such as cyclosporin, azathioprine,methotrexate, mycophenolate, and FK506, antibodies, or otherimmunoablative agents such as CAMPATH, anti-CD3 antibodies, cytoxan,fludarabine, cyclosporin, FK506, rapamycin, mycophenolic acid, steroids,FR901228, and irradiation. These drugs inhibit either the calciumdependent phosphatase calcineurin (cyclosporine and FK506) or inhibitthe p70S6 kinase that is important for growth factor induced signaling(rapamycin). (Liu et al., Cell 66:807-815, 1991; Henderson et al.,Immun. 73:316-321, 1991; Bierer et al., Curr. Opin. Immun. 5:763-773,1993). In a further aspect, the cells are isolated for a patient andfrozen for later use in conjunction with (e.g., before, simultaneouslyor following) bone marrow or stem cell transplantation, T cell ablativetherapy using either chemotherapy agents such as, fludarabine,external-beam radiation therapy (XRT), cyclophosphamide, or antibodiessuch as OKT3 or CAMPATH. In one aspect, the cells are isolated prior toand can be frozen for later use for treatment following B-cell ablativetherapy such as agents that react with CD20, e.g., Rituxan.

In a further aspect of the present invention, T cells are obtained froma patient directly following treatment that leaves the subject withfunctional T cells. In this regard, it has been observed that followingcertain cancer treatments, in particular treatments with drugs thatdamage the immune system, shortly after treatment during the period whenpatients would normally be recovering from the treatment, the quality ofT cells obtained may be optimal or improved for their ability to expandex vivo. Likewise, following ex vivo manipulation using the methodsdescribed herein, these cells may be in a preferred state for enhancedengraftment and in vivo expansion. Thus, it is contemplated within thecontext of the invention to collect blood cells, including T cells,dendritic cells, or other cells of the hematopoietic lineage, duringthis recovery phase. Further, in certain aspects, mobilization (forexample, mobilization with GM-CSF) and conditioning regimens can be usedto create a condition in a subject wherein repopulation, recirculation,regeneration, and/or expansion of particular cell types is favored,especially during a defined window of time following therapy.Illustrative cell types include T cells, B cells, dendritic cells, andother cells of the immune system.

In an embodiment, the NK cells are obtained from the subject. In anotherembodiment, the NK cells are an NK cell line, e.g., NK-92 cell line(Conkwest).

Allogeneic CAR

In embodiments described herein, the immune effector cell can be anallogeneic immune effector cell, e.g., T cell or NK cell. For example,the cell can be an allogeneic T cell, e.g., an allogeneic T cell lackingexpression of a functional T cell receptor (TCR) and/or human leukocyteantigen (HLA), e.g., HLA class I and/or HLA class II.

A T cell lacking a functional TCR can be, e.g., engineered such that itdoes not express any functional TCR on its surface, engineered such thatit does not express one or more subunits that comprise a functional TCRor engineered such that it produces very little functional TCR on itssurface. Alternatively, the T cell can express a substantially impairedTCR, e.g., by expression of mutated or truncated forms of one or more ofthe subunits of the TCR. The term “substantially impaired TCR” meansthat this TCR will not elicit an adverse immune reaction in a host.

A T cell described herein can be, e.g., engineered such that it does notexpress a functional HLA on its surface. For example, a T cell describedherein, can be engineered such that cell surface expression HLA, e.g.,HLA class 1 and/or HLA class II, is downregulated.

In some embodiments, the T cell can lack a functional TCR and afunctional HLA, e.g., HLA class I and/or HLA class II.

Modified T cells that lack expression of a functional TCR and/or HLA canbe obtained by any suitable means, including a knock out or knock downof one or more subunit of TCR or HLA. For example, the T cell caninclude a knock down of TCR and/or HLA using siRNA, shRNA, clusteredregularly interspaced short palindromic repeats (CRISPR)transcription-activator like effector nuclease (TALEN), or zinc fingerendonuclease (ZFN).

In some embodiments, the allogeneic cell can be a cell which does notexpresses or expresses at low levels an inhibitory molecule, e.g. by anymehod described herein. For example, the cell can be a cell that doesnot express or expresses at low levels an inhibitory molecule, e.g.,that can decrease the ability of a CAR-expressing cell to mount animmune effector response. Examples of inhibitory molecules include PD1,PD-L1, CTLA4, TIM3, CEACAM (e.g., CEACAM-1, CEACAM-3 and/or CEACAM-5),LAG3, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4 and TGFR beta. Inhibition ofan inhibitory molecule, e.g., by inhibition at the DNA, RNA or proteinlevel, can optimize a CAR-expressing cell performance. In embodiments,an inhibitory nucleic acid, e.g., an inhibitory nucleic acid, e.g., adsRNA, e.g., an siRNA or shRNA, a clustered regularly interspaced shortpalindromic repeats (CRISPR), a transcription-activator like effectornuclease (TALEN), or a zinc finger endonuclease (ZFN), e.g., asdescribed herein, can be used.

siRNA and shRNA to Inhibit TCR or HLA

In some embodiments, TCR expression and/or HLA expression can beinhibited using siRNA or shRNA that targets a nucleic acid encoding aTCR and/or HLA in a T cell.

Expression of siRNA and shRNAs in T cells can be achieved using anyconventional expression system, e.g., such as a lentiviral expressionsystem.

Exemplary shRNAs that downregulate expression of components of the TCRare described, e.g., in US Publication No.: 2012/0321667. ExemplarysiRNA and shRNA that downregulate expression of HLA class I and/or HLAclass II genes are described, e.g., in U.S. publication No.: US2007/0036773.

CRISPR to inhibit TCR or HLA

“CRISPR” or “CRISPR to TCR and/or HLA” or “CRISPR to inhibit TCR and/orHLA” as used herein refers to a set of clustered regularly interspacedshort palindromic repeats, or a system comprising such a set of repeats.“Cas”, as used herein, refers to a CRISPR-associated protein. A“CRISPR/Cas” system refers to a system derived from CRISPR and Cas whichcan be used to silence or mutate a TCR and/or HLA gene.

Naturally-occurring CRISPR/Cas systems are found in approximately 40% ofsequenced eubacteria genomes and 90% of sequenced archaea. Grissa et al.(2007) BMC Bioinformatics 8: 172. This system is a type of prokaryoticimmune system that confers resistance to foreign genetic elements suchas plasmids and phages and provides a form of acquired immunity.Barrangou et al. (2007) Science 315: 1709-1712; Marragini et al. (2008)Science 322: 1843-1845.

The CRISPR/Cas system has been modified for use in gene editing(silencing, enhancing or changing specific genes) in eukaryotes such asmice or primates. Wiedenheft et al. (2012) Nature 482: 331-8. This isaccomplished by introducing into the eukaryotic cell a plasmidcontaining a specifically designed CRISPR and one or more appropriateCas.

The CRISPR sequence, sometimes called a CRISPR locus, comprisesalternating repeats and spacers. In a naturally-occurring CRISPR, thespacers usually comprise sequences foreign to the bacterium such as aplasmid or phage sequence; in the TCR and/or HLA CRISPR/Cas system, thespacers are derived from the TCR or HLA gene sequence.

RNA from the CRISPR locus is constitutively expressed and processed byCas proteins into small RNAs. These comprise a spacer flanked by arepeat sequence. The RNAs guide other Cas proteins to silence exogenousgenetic elements at the RNA or DNA level. Horvath et al. (2010) Science327: 167-170; Makarova et al. (2006) Biology Direct 1: 7. The spacersthus serve as templates for RNA molecules, analogously to siRNAs.Pennisi (2013) Science 341: 833-836.

As these naturally occur in many different types of bacteria, the exactarrangements of the CRISPR and structure, function and number of Casgenes and their product differ somewhat from species to species. Haft etal. (2005) PLoS Comput. Biol. 1: e60; Kunin et al. (2007) Genome Biol.8: R61; Mojica et al. (2005) J Mol. Evol. 60: 174-182; Bolotin et al.(2005) Microbiol. 151: 2551-2561; Pourcel et al. (2005) Microbiol. 151:653-663; and Stern et al. (2010) Trends. Genet. 28: 335-340. Forexample, the Cse (Cas subtype, E. coli) proteins (e.g., CasA) form afunctional complex, Cascade, that processes CRISPR RNA transcripts intospacer-repeat units that Cascade retains. Brouns et al. (2008) Science321: 960-964. In other prokaryotes, Cas6 processes the CRISPRtranscript. The CRISPR-based phage inactivation in E. coli requiresCascade and Cas3, but not Cas1 or Cas2. The Cmr (Cas RAMP module)proteins in Pyrococcus furiosus and other prokaryotes form a functionalcomplex with small CRISPR RNAs that recognizes and cleaves complementarytarget RNAs. A simpler CRISPR system relies on the protein Cas9, whichis a nuclease with two active cutting sites, one for each strand of thedouble helix. Combining Cas9 and modified CRISPR locus RNA can be usedin a system for gene editing. Pennisi (2013) Science 341: 833-836.

The CRISPR/Cas system can thus be used to edit a TCR and/or HLA gene(adding or deleting a basepair), or introducing a premature stop whichthus decreases expression of a TCR and/or HLA. The CRISPR/Cas system canalternatively be used like RNA interference, turning off TCR and/or HLAgene in a reversible fashion. In a mammalian cell, for example, the RNAcan guide the Cas protein to a TCR and/or HLA promoter, stericallyblocking RNA polymerases.

Artificial CRISPR/Cas systems can be generated which inhibit TCR and/orHLA, using technology known in the art, e.g., that described in U.S.Publication No.20140068797, and Cong (2013) Science 339: 819-823. Otherartificial CRISPR/Cas systems that are known in the art may also begenerated which inhibit TCR and/or HLA, e.g., that described in Tsai(2014) Nature Biotechnol., 32:6 569-576, U.S. Pat. Nos. 8,871,445;8,865,406; 8,795,965; 8,771,945; and 8,697,359.

TALEN to Inhibit TCR and/or HLA

“TALEN” or “TALEN to HLA and/or TCR” or “TALEN to inhibit HLA and/orTCR” refers to a transcription activator-like effector nuclease, anartificial nuclease which can be used to edit the HLA and/or TCR gene.

TALENs are produced artificially by fusing a TAL effector DNA bindingdomain to a DNA cleavage domain. Transcription activator-like effects(TALEs) can be engineered to bind any desired DNA sequence, including aportion of the HLA or TCR gene. By combining an engineered TALE with aDNA cleavage domain, a restriction enzyme can be produced which isspecific to any desired DNA sequence, including a HLA or TCR sequence.These can then be introduced into a cell, wherein they can be used forgenome editing. Boch (2011) Nature Biotech. 29: 135-6; and Boch et al.(2009) Science 326: 1509-12; Moscou et al. (2009) Science 326: 3501.

TALEs are proteins secreted by Xanthomonas bacteria. The DNA bindingdomain contains a repeated, highly conserved 33-34 amino acid sequence,with the exception of the 12th and 13th amino acids. These two positionsare highly variable, showing a strong correlation with specificnucleotide recognition. They can thus be engineered to bind to a desiredDNA sequence.

To produce a TALEN, a TALE protein is fused to a nuclease (N), which isa wild-type or mutated FokI endonuclease. Several mutations to FokI havebeen made for its use in TALENs; these, for example, improve cleavagespecificity or activity. Cermak et al. (2011) Nucl. Acids Res. 39: e82;Miller et al. (2011) Nature Biotech. 29: 143-8; Hockemeyer et al. (2011)Nature Biotech. 29: 731-734; Wood et al. (2011) Science 333: 307; Doyonet al. (2010) Nature Methods 8: 74-79; Szczepek et al. (2007) NatureBiotech. 25: 786-793; and Guo et al. (2010)J Mol. Biol. 200: 96.

The FokI domain functions as a dimer, requiring two constructs withunique DNA binding domains for sites in the target genome with properorientation and spacing. Both the number of amino acid residues betweenthe TALE DNA binding domain and the FokI cleavage domain and the numberof bases between the two individual TALEN binding sites appear to beimportant parameters for achieving high levels of activity. Miller etal. (2011) Nature Biotech. 29: 143-8.

A HLA or TCR TALEN can be used inside a cell to produce adouble-stranded break (DSB). A mutation can be introduced at the breaksite if the repair mechanisms improperly repair the break vianon-homologous end joining. For example, improper repair may introduce aframe shift mutation. Alternatively, foreign DNA can be introduced intothe cell along with the TALEN; depending on the sequences of the foreignDNA and chromosomal sequence, this process can be used to correct adefect in the HLA or TCR gene or introduce such a defect into a wt HLAor TCR gene, thus decreasing expression of HLA or TCR.

TALENs specific to sequences in HLA or TCR can be constructed using anymethod known in the art, including various schemes using modularcomponents. Zhang et al. (2011) Nature Biotech. 29: 149-53; Geibler etal. (2011) PLoS ONE 6: e19509.

Zinc Finger Nuclease to Inhibit HLA and/or TCR

“ZFN” or “Zinc Finger Nuclease” or “ZFN to HLA and/or TCR” or “ZFN toinhibit HLA and/or TCR” refer to a zinc finger nuclease, an artificialnuclease which can be used to edit the HLA and/or TCR gene.

Like a TALEN, a ZFN comprises a FokI nuclease domain (or derivativethereof) fused to a DNA-binding domain. In the case of a ZFN, theDNA-binding domain comprises one or more zinc fingers. Carroll et al.(2011) Genetics Society of America 188: 773-782; and Kim et al. (1996)Proc. Natl. Acad. Sci. USA 93: 1156-1160.

A zinc finger is a small protein structural motif stabilized by one ormore zinc ions. A zinc finger can comprise, for example, Cys2His2, andcan recognize an approximately 3-bp sequence. Various zinc fingers ofknown specificity can be combined to produce multi-finger polypeptideswhich recognize about 6, 9, 12, 15 or 18-bp sequences. Various selectionand modular assembly techniques are available to generate zinc fingers(and combinations thereof) recognizing specific sequences, includingphage display, yeast one-hybrid systems, bacterial one-hybrid andtwo-hybrid systems, and mammalian cells.

Like a TALEN, a ZFN must dimerize to cleave DNA. Thus, a pair of ZFNsare required to target non-palindromic DNA sites. The two individualZFNs must bind opposite strands of the DNA with their nucleases properlyspaced apart. Bitinaite et al. (1998) Proc. Natl. Acad. Sci. USA 95:10570-5.

Also like a TALEN, a ZFN can create a double-stranded break in the DNA,which can create a frame-shift mutation if improperly repaired, leadingto a decrease in the expression and amount of HLA and/or TCR in a cell.ZFNs can also be used with homologous recombination to mutate in the HLAor TCR gene.

ZFNs specific to sequences in HLA AND/OR TCR can be constructed usingany method known in the art. See, e.g., Provasi (2011) Nature Med. 18:807-815; Torikai (2013) Blood 122: 1341-1349; Cathomen et al. (2008)Mol. Ther. 16: 1200-7; and Guo et al. (2010) J. Mol. Biol. 400: 96; U.S.Patent Publication 2011/0158957; U.S. Patent Publication 2012/0060230.

Activation and Expansion of Cells

Cells may be activated and expanded generally using methods asdescribed, for example, in U.S. Pat. Nos. 6,352,694; 6,534,055;6,905,680; 6,692,964; 5,858,358; 6,887,466; 6,905,681; 7,144,575;7,067,318; 7,172,869; 7,232,566; 7,175,843; 5,883,223; 6,905,874;6,797,514; 6,867,041; and U.S. Patent Application Publication No.20060121005.

Generally, the T cells of the invention may be expanded by contact witha surface having attached thereto an agent that stimulates a CD3/TCRcomplex associated signal and a ligand that stimulates a costimulatorymolecule on the surface of the T cells. In particular, T cellpopulations may be stimulated as described herein, such as by contactwith an anti-CD3 antibody, or antigen-binding fragment thereof, or ananti-CD2 antibody immobilized on a surface, or by contact with a proteinkinase C activator (e.g., bryostatin) in conjunction with a calciumionophore. For co-stimulation of an accessory molecule on the surface ofthe T cells, a ligand that binds the accessory molecule is used. Forexample, a population of T cells can be contacted with an anti-CD3antibody and an anti-CD28 antibody, under conditions appropriate forstimulating proliferation of the T cells. To stimulate proliferation ofeither CD4+ T cells or CD8+ T cells, an anti-CD3 antibody and ananti-CD28 antibody. Examples of an anti-CD28 antibody include 9.3, B-T3,XR-CD28 (Diaclone, Besancon, France) can be used as can other methodscommonly known in the art (Berg et al., Transplant Proc.30(8):3975-3977, 1998; Haanen et al., J. Exp. Med. 190(9):13191328,1999; Garland et al., J. Immunol Meth. 227(1-2):53-63, 1999).

In certain aspects, the primary stimulatory signal and the costimulatorysignal for the T cell may be provided by different protocols. Forexample, the agents providing each signal may be in solution or coupledto a surface. When coupled to a surface, the agents may be coupled tothe same surface (i.e., in “cis” formation) or to separate surfaces(i.e., in “trans” formation). Alternatively, one agent may be coupled toa surface and the other agent in solution. In one aspect, the agentproviding the costimulatory signal is bound to a cell surface and theagent providing the primary activation signal is in solution or coupledto a surface. In certain aspects, both agents can be in solution. In oneaspect, the agents may be in soluble form, and then cross-linked to asurface, such as a cell expressing Fc receptors or an antibody or otherbinding agent which will bind to the agents. In this regard, see forexample, U.S. Patent Application Publication Nos. 20040101519 and20060034810 for artificial antigen presenting cells (aAPCs) that arecontemplated for use in activating and expanding T cells in the presentinvention.

In one aspect, the two agents are immobilized on beads, either on thesame bead, i.e., “cis,” or to separate beads, i.e., “trans.” By way ofexample, the agent providing the primary activation signal is ananti-CD3 antibody or an antigen-binding fragment thereof and the agentproviding the costimulatory signal is an anti-CD28 antibody orantigen-binding fragment thereof; and both agents are co-immobilized tothe same bead in equivalent molecular amounts. In one aspect, a 1:1ratio of each antibody bound to the beads for CD4+ T cell expansion andT cell growth is used. In certain aspects of the present invention, aratio of anti CD3:CD28 antibodies bound to the beads is used such thatan increase in T cell expansion is observed as compared to the expansionobserved using a ratio of 1:1. In one particular aspect an increase offrom about 1 to about 3 fold is observed as compared to the expansionobserved using a ratio of 1:1. In one aspect, the ratio of CD3:CD28antibody bound to the beads ranges from 100:1 to 1:100 and all integervalues there between. In one aspect of the present invention, moreanti-CD28 antibody is bound to the particles than anti-CD3 antibody,i.e., the ratio of CD3:CD28 is less than one. In certain aspects of theinvention, the ratio of anti CD28 antibody to anti CD3 antibody bound tothe beads is greater than 2:1. In one particular aspect, a 1:100CD3:CD28 ratio of antibody bound to beads is used. In one aspect, a 1:75CD3:CD28 ratio of antibody bound to beads is used. In a further aspect,a 1:50 CD3:CD28 ratio of antibody bound to beads is used. In one aspect,a 1:30 CD3:CD28 ratio of antibody bound to beads is used. In onepreferred aspect, a 1:10 CD3:CD28 ratio of antibody bound to beads isused. In one aspect, a 1:3 CD3:CD28 ratio of antibody bound to the beadsis used. In yet one aspect, a 3:1 CD3:CD28 ratio of antibody bound tothe beads is used.

Ratios of particles to cells from 1:500 to 500:1 and any integer valuesin between may be used to stimulate T cells or other target cells. Asthose of ordinary skill in the art can readily appreciate, the ratio ofparticles to cells may depend on particle size relative to the targetcell. For example, small sized beads could only bind a few cells, whilelarger beads could bind many. In certain aspects the ratio of cells toparticles ranges from 1:100 to 100:1 and any integer values in-betweenand in further aspects the ratio comprises 1:9 to 9:1 and any integervalues in between, can also be used to stimulate T cells. The ratio ofanti-CD3- and anti-CD28-coupled particles to T cells that result in Tcell stimulation can vary as noted above, however certain preferredvalues include 1:100, 1:50, 1:40, 1:30, 1:20, 1:10, 1:9, 1:8, 1:7, 1:6,1:5, 1:4, 1:3, 1:2, 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1,and 15:1 with one preferred ratio being at least 1:1 particles per Tcell. In one aspect, a ratio of particles to cells of 1:1 or less isused. In one particular aspect, a preferred particle:cell ratio is 1:5.In further aspects, the ratio of particles to cells can be varieddepending on the day of stimulation. For example, in one aspect, theratio of particles to cells is from 1:1 to 10:1 on the first day andadditional particles are added to the cells every day or every other daythereafter for up to 10 days, at final ratios of from 1:1 to 1:10 (basedon cell counts on the day of addition). In one particular aspect, theratio of particles to cells is 1:1 on the first day of stimulation andadjusted to 1:5 on the third and fifth days of stimulation. In oneaspect, particles are added on a daily or every other day basis to afinal ratio of 1:1 on the first day, and 1:5 on the third and fifth daysof stimulation. In one aspect, the ratio of particles to cells is 2:1 onthe first day of stimulation and adjusted to 1:10 on the third and fifthdays of stimulation. In one aspect, particles are added on a daily orevery other day basis to a final ratio of 1:1 on the first day, and 1:10on the third and fifth days of stimulation. One of skill in the art willappreciate that a variety of other ratios may be suitable for use in thepresent invention. In particular, ratios will vary depending on particlesize and on cell size and type. In one aspect, the most typical ratiosfor use are in the neighborhood of 1:1, 2:1 and 3:1 on the first day.

In further aspects of the present invention, the cells, such as T cells,are combined with agent-coated beads, the beads and the cells aresubsequently separated, and then the cells are cultured. In analternative aspect, prior to culture, the agent-coated beads and cellsare not separated but are cultured together. In a further aspect, thebeads and cells are first concentrated by application of a force, suchas a magnetic force, resulting in increased ligation of cell surfacemarkers, thereby inducing cell stimulation.

By way of example, cell surface proteins may be ligated by allowingparamagnetic beads to which anti-CD3 and anti-CD28 are attached (3×28beads) to contact the T cells. In one aspect the cells (for example, 10⁴to 10⁹ T cells) and beads (for example, DYNABEADS® M-450 CD3/CD28 Tparamagnetic beads at a ratio of 1:1) are combined in a buffer, forexample PBS (without divalent cations such as, calcium and magnesium).Again, those of ordinary skill in the art can readily appreciate anycell concentration may be used. For example, the target cell may be veryrare in the sample and comprise only 0.01% of the sample or the entiresample (i.e., 100%) may comprise the target cell of interest.Accordingly, any cell number is within the context of the presentinvention. In certain aspects, it may be desirable to significantlydecrease the volume in which particles and cells are mixed together(i.e., increase the concentration of cells), to ensure maximum contactof cells and particles. For example, in one aspect, a concentration ofabout 2 billion cells/ml is used. In one aspect, greater than 100million cells/ml is used. In a further aspect, a concentration of cellsof 10, 15, 20, 25, 30, 35, 40, 45, or 50 million cells/ml is used. Inyet one aspect, a concentration of cells from 75, 80, 85, 90, 95, or 100million cells/ml is used. In further aspects, concentrations of 125 or150 million cells/ml can be used. Using high concentrations can resultin increased cell yield, cell activation, and cell expansion. Further,use of high cell concentrations allows more efficient capture of cellsthat may weakly express target antigens of interest, such asCD28-negative T cells. Such populations of cells may have therapeuticvalue and would be desirable to obtain in certain aspects. For example,using high concentration of cells allows more efficient selection ofCD8+ T cells that normally have weaker CD28 expression.

In one aspect of the present invention, the mixture may be cultured forseveral hours (about 3 hours) to about 14 days or any hourly integervalue in between. In one aspect, the mixture may be cultured for 21days. In one aspect of the invention the beads and the T cells arecultured together for about eight days. In one aspect, the beads and Tcells are cultured together for 2-3 days. Several cycles of stimulationmay also be desired such that culture time of T cells can be 60 days ormore. Conditions appropriate for T cell culture include an appropriatemedia (e.g., Minimal Essential Media or RPMI Media 1640 or, X-vivo 15,(Lonza)) that may contain factors necessary for proliferation andviability, including serum (e.g., fetal bovine or human serum),interleukin-2 (IL-2), insulin, IFN-γ, IL-4, IL-7, GM-CSF, IL-10, IL-12,IL-15, TGFβ, and TNF-α or any other additives for the growth of cellsknown to the skilled artisan. Other additives for the growth of cellsinclude, but are not limited to, surfactant, plasmanate, and reducingagents such as N-acetyl-cysteine and 2-mercaptoethanol. Media caninclude RPMI 1640, AIM-V, DMEM, MEM, α-MEM, F-12, X-Vivo 15, and X-Vivo20, Optimizer, with added amino acids, sodium pyruvate, and vitamins,either serum-free or supplemented with an appropriate amount of serum(or plasma) or a defined set of hormones, and/or an amount ofcytokine(s) sufficient for the growth and expansion of T cells.Antibiotics, e.g., penicillin and streptomycin, are included only inexperimental cultures, not in cultures of cells that are to be infusedinto a subject. The target cells are maintained under conditionsnecessary to support growth, for example, an appropriate temperature(e.g., 37° C.) and atmosphere (e.g., air plus 5% CO₂).

T cells that have been exposed to varied stimulation times may exhibitdifferent characteristics. For example, typical blood or apheresedperipheral blood mononuclear cell products have a helper T cellpopulation (TH, CD4+) that is greater than the cytotoxic or suppressor Tcell population (TC, CD8+). Ex vivo expansion of T cells by stimulatingCD3 and CD28 receptors produces a population of T cells that prior toabout days 8-9 consists predominately of TH cells, while after aboutdays 8-9, the population of T cells comprises an increasingly greaterpopulation of TC cells. Accordingly, depending on the purpose oftreatment, infusing a subject with a T cell population comprisingpredominately of TH cells may be advantageous. Similarly, if anantigen-specific subset of TC cells has been isolated it may bebeneficial to expand this subset to a greater degree.

Further, in addition to CD4 and CD8 markers, other phenotypic markersvary significantly, but in large part, reproducibly during the course ofthe cell expansion process. Thus, such reproducibility enables theability to tailor an activated T cell product for specific purposes.

Once a mesothelin CAR is constructed, various assays can be used toevaluate the activity of the molecule, such as but not limited to, theability to expand T cells following antigen stimulation, sustain T cellexpansion in the absence of re-stimulation, and anti-cancer activitiesin appropriate in vitro and animal models. Assays to evaluate theeffects of a mesothelin CAR are described in further detail below

Western blot analysis of CAR expression in primary T cells can be usedto detect the presence of monomers and dimers. See, e.g., Milone et al.,Molecular Therapy 17(8): 1453-1464 (2009). Very briefly, T cells (1:1mixture of CD4⁺ and CD8⁺ T cells) expressing the CARs are expanded invitro for more than 10 days followed by lysis and SDS-PAGE underreducing conditions. CARs containing the full length TCR-ζ cytoplasmicdomain and the endogenous TCR-ζ chain are detected by western blottingusing an antibody to the TCR-ζ chain. The same T cell subsets are usedfor SDS-PAGE analysis under non-reducing conditions to permit evaluationof covalent dimer formation.

In vitro expansion of CAR′ T cells following antigen stimulation can bemeasured by flow cytometry. For example, a mixture of CD4⁺ and CD8⁺ Tcells are stimulated with αCD3/αCD28 aAPCs followed by transduction withlentiviral vectors expressing GFP under the control of the promoters tobe analyzed. Exemplary promoters include the CMV IE gene, EF-1α,ubiquitin C, or phosphoglycerokinase (PGK) promoters. GFP fluorescenceis evaluated on day 6 of culture in the CD4⁺ and/or CD8⁺ T cell subsetsby flow cytometry. See, e.g., Milone et al., Molecular Therapy 17(8):1453-1464 (2009). Alternatively, a mixture of CD4⁺ and CD8⁺ T cells arestimulated with αCD3/αCD28 coated magnetic beads on day 0, andtransduced with CAR on day 1 using a bicistronic lentiviral vectorexpressing CAR along with eGFP using a 2A ribosomal skipping sequence.Cultures are re-stimulated, e.g., with K562 cells expressing hCD32 and4-1BBL in the presence of anti-CD3 and anti-CD28 antibody(K562-BBL-3/28) following washing. Exogenous IL-2 is added to thecultures every other day at 100 IU/ml. GFP⁺ T cells are enumerated byflow cytometry using bead-based counting. See, e.g., Milone et al.,Molecular Therapy 17(8): 1453-1464 (2009).

Sustained CAR⁺ T cell expansion in the absence of re-stimulation canalso be measured. See, e.g., Milone et al., Molecular Therapy 17(8):1453-1464 (2009). Briefly, mean T cell volume (f1) is measured on day 8of culture using a Coulter Multisizer III particle counter followingstimulation with αCD3/αCD28 coated magnetic beads on day 0, andtransduction with the indicated CAR on day 1.

Assessment of cell proliferation and cytokine production has beenpreviously described, e.g., at Milone et al., Molecular Therapy 17(8):1453-1464 (2009). Briefly, assessment of CAR-mediated proliferation isperformed in microtiter plates by mixing washed T cells with targetcells, such as K562-Meso, Ovcar3, Ovcar8, SW1990, Panc02.03 cellsexpressing mesothelin or CD32 and CD137 (KT32-BBL) for a finalT-cell:target cell ratio of 1:1. Anti-CD3 (clone OKT3) and anti-CD28(clone 9.3) monoclonal antibodies are added to cultures with KT32-BBLcells to serve as a positive control for stimulating T-cellproliferation since these signals support long-term CD8⁺ T cellexpansion ex vivo. T cells are enumerated in cultures using CountBright™fluorescent beads (Invitrogen, Carlsbad, Calif.) and flow cytometry asdescribed by the manufacturer. CAR⁺ T cells are identified by GFPexpression using T cells that are engineered with eGFP-2A linkedCAR-expressing lentiviral vectors. For CAR+ T cells not expressing GFP,the CAR+ T cells are detected with biotinylated recombinant mesothelinprotein and a secondary avidin-PE conjugate. CD4+ and CD8⁺ expression onT cells are also simultaneously detected with specific monoclonalantibodies (BD Biosciences). Cytokine measurements are performed onsupernatants collected 24 hours following re-stimulation using the humanTH1/TH2 cytokine cytometric bead array kit (BD Biosciences, San Diego,Calif.) according the manufacturer's instructions. Fluorescence isassessed using a FACScalibur flow cytometer, and data is analyzedaccording to the manufacturer's instructions.

Cytotoxicity can be assessed by methods described herein, e.g., in theexamples, or by a standard 51Cr-release assay. See, e.g., Milone et al.,Molecular Therapy 17(8): 1453-1464 (2009). Briefly, target cells (e.g.,BHK or CHO cells expressing mesothelin) are loaded with 51Cr (as NaCrO4,New England Nuclear, Boston, Mass.) at 37° C. for 2 hours with frequentagitation, washed twice in complete RPMI and plated into microtiterplates. Effector T cells are mixed with target cells in the wells incomplete RPMI at varying ratios of effector cell:target cell (E:T).Additional wells containing media only (spontaneous release, SR) or a 1%solution of triton-X 100 detergent (total release, TR) are alsoprepared. After 4 hours of incubation at 37° C., supernatant from eachwell is harvested. Released ⁵¹Cr is then measured using a gamma particlecounter (Packard Instrument Co., Waltham, Mass.). Each condition isperformed in at least triplicate, and the percentage of lysis iscalculated using the formula: % Lysis=(ER−SR)/(TR−SR), where ERrepresents the average 51Cr released for each experimental condition.Alternative cytotoxicity assays may also be used, such as flow basedcytotoxicity assays.

Click beetle red and click beetle green luciferase can be used tosimultaneously follow tumor progression and T cell trafficking, as eachuse the same luciferin substrate but emit light at the opposite ends ofthe visible light spectrum.

Other assays, including those described in the Example section herein aswell as those that are known in the art can also be used to evaluate themesothelin CAR constructs of the invention.

Therapeutic Application for Mesothelin Expressing Diseases and Disorders

The present invention provides compositions and methods for treatingdiseases and disorders associated with mesothelin. An example of adisease or disorder associated with mesothelin is mesothelioma.

Malignant mesothelioma is a type of cancer that occurs in the thin layerof cells lining the body's internal organs, known as the mesothelium.There are three recognized types of mesothelioma. Pleural mesothelioma(e.g., malignant pleural mesothelioma, or MPM) is the most common formof the disease, accounting for roughly 70% of cases, and occurs in thelining of the lung known as the pleura. Peritoneal mesothelioma occursin the lining of the abdominal cavity, known as the peritoneum.Pericardial mesothelioma originates in the pericardium, which lines theheart.

A subject may be at risk to develop mesothelioma if the subject wasexposed to asbestos. Exposure to asbestos and the inhalation of asbestosparticles can cause mesothelioma. In most cases, mesothelioma symptomswill not appear in a subject exposed to asbestos until many years afterthe exposure has occurred.

Symptoms of pleural mesothelioma include, e.g., lower back pain or sidechest pain, and shortness of breath. Other symptoms include difficultyswallowing, persistent cough, fever, weight loss or fatigue. Additionalsymptoms that some patients experience are muscle weakness, loss ofsensory capability, coughing up blood, facial and arm swelling, andhoarseness. In the early stages of the disease, such as stage 1mesothelioma, symptoms may be mild. Patients usually report pain in onearea of the chest that never seems to go away, weight loss and fever.

Peritoneal mesothelioma originates in the abdomen and as a result,symptoms often include abdominal pain, weight loss, nausea, andvomiting. Fluid buildup may occur in the abdomen as well as a result ofthe cancer. Peritoneal mesothelioma originates in the abdomen and willfrequently spread to other organs in area including the liver, spleen orbowel. Severe abdominal pain is the most common complaint that patientsfirst experience. There may also be a discomfort level with fluidbuildup in the abdomen as well. Other symptoms of peritonealmesothelioma may include difficult bowel movements, nausea and vomiting,fever and swollen feet.

Pericardial mesothelioma is the least common form of mesothelioma.Pericardial mesothelioma, as the name suggests, involves the heart. Thisrare type of mesothelioma cancer invades the pericardium, the sac thatsurrounds the heart. As the cancer progresses, the heart is not able todeliver oxygen as efficiently to the body causing further decline inhealth at an increasingly rapid rate. The symptoms most commonlyassociated with pericardial mesothelioma mimic those of a heart attack:nausea, pain in the chest and shortness of breath.

Subjects benefiting from treatment according to the invention includesubjects with a mesothelioma, or subjects suspected of havingmesothelioma, e.g., as evidenced by the presence of one or more of thesymptoms described herein and/or exposure to asbestos. In particularembodiments, the mesothelioma is pleural mesothelioma (e.g., malignantpleural mesothelioma). In other aspects, the subject may be treated thathas a precancerous condition such as, e.g., pleural plaques, benignmesothelioma or mesothelial hyperplasia.

Another example of a disease or disorder associated with mesothelin ispancreatic cancer. Pancreatic cancers that can be treated with methodsdescribed herein include, but are not limited to, exocrine pancreaticcancers and endocrine pancreatic cancers. Exocrine pancreatic cancersinclude, but are not limited to, adenocarcinomas, acinar cellcarcinomas, adenosquamous carcinomas, colloid carcinomas,undifferentiated carcinomas with osteoclast-like giant cells, hepatoidcarcinomas, intraductal papillary-mucinous neoplasms, mucinous cysticneoplasms, pancreatoblastomas, serous cystadenomas, signet ring cellcarcinomas, solid and pseuodpapillary tumors, pancreatic ductalcarcinomas, and undifferentiated carcinomas. In some embodiments, theexocrine pancreatic cancer is pancreatic ductal carcinoma. Endocrinepancreatic cancers include, but are not limited to, insulinomas andglucagonomas.

In some embodiments, the pancreatic cancer is any of early stagepancreatic cancer, non-metastatic pancreatic cancer, primary pancreaticcancer, resected pancreatic cancer, advanced pancreatic cancer, locallyadvanced pancreatic cancer, metastatic pancreatic cancer, unresectablepancreatic cancer, pancreatic cancer in remission, recurrent pancreaticcancer, pancreatic cancer in an adjuvant setting, or pancreatic cancerin a neoadjuvant setting. In some embodiments, the pancreatic cancer islocally advanced pancreatic cancer, unresectable pancreatic cancer, ormetastatic pancreatic ductal carcinoma. In some embodiments, thepancreatic cancer is resistant to the gemcitabine-based therapy. In someembodiments, the pancreatic cancer is refractory to thegemcitabine-based therapy.

In other aspects, the disorder associated with mesothelin expression isovarian cancer. Ovarian cancer is classified according to the histologyof the tumor. Surface epithelial-stromal tumor, also known as ovarianepithelial carcinoma, is the most common type of ovarian cancer. Itincludes serous tumor (including serous papillary cystadenocarcinoma),endometrioid tumor and mucinous cystadenocarcinoma.

The methods described herein can be used to treat various stages ofovarian cancer, e.g., stage I, stage II, stage III or stage IV. Stagingcan be performed, e.g., when the ovarian cancer is removed. Ovariancancer is staged as follows:

Stage I cancer is confined to one or both ovaries. The cancer is stageII if either one or both of the ovaries is involved and has spread tothe uterus and/or the fallopian tubes or other sites in the pelvis. Thecancer is stage III cancer if one or both of the ovaries is involved andhas spread to lymph nodes or other sites outside of the pelvis but isstill within the abdominal cavity, such as the surface of the intestineor liver. The cancer is stage IV cancer if one or both ovaries areinvolved and the cancer has spread outside the abdomen or to the insideof the liver.

In some embodiments, the ovarian cancer is resistant to one or morechemotherapeutic agent. In some embodiments, the ovarian cancer isrefractory to the one or more chemotherapeutic agent.

Other cancers that can be treated with the CAR compositions describedherein include, e.g., brain cancer, bladder cancer, breast cancer,cervical cancer, colorectal cancer, liver cancer, kidney cancer,lymphoma, leukemia, lung cancer (e.g., lung adenocarcinoma), melanoma,metastatic melanoma, mesothelioma, neuroblastoma, ovarian cancer,prostate cancer, pancreatic cancer, renal cancer, skin cancer, thymoma,sarcoma, non-Hodgkin's lymphoma, Hodgkin's lymphoma, uterine cancer, andany combination thereof.

The present invention provides methods for inhibiting the proliferationor reducing a mesothelin-expressing cell population, the methodscomprising contacting a population of cells comprising a mesothelinexpressing cell with a mesothelin CAR-expressing cell of the inventionthat binds to the mesothelin-expressing cell. In a specific embodiment,the invention provides methods for inhibiting the proliferation orreducing the population of cancer cells expressing mesothelin, themethods comprising contacting the mesothelin-expressing cancer cellpopulation with a mesothelin CAR-expressing cell of the invention thatbinds to the mesothelin-expressing cell. In another embodiment, theinvention provides methods for inhibiting the proliferation or reducingthe population of cancer cells expressing mesothelin, the methodscomprising contacting the mesothelin-expressing cancer cell populationwith a mesothelin CAR-expressing cell of the invention that binds to themesothelin-expressing cell. In certain embodiments, the mesothelinCAR-expressing cell of the invention reduces the quantity, number,amount or percentage of cells and/or cancer cells by at least 25%, atleast 30%, at least 40%, at least 50%, at least 65%, at least 75%, atleast 85%, at least 95%, or at least 99% in a subject with or animalmodel of mesothelioma or another cancer associated withmesothelin-expressing cells relative to a negative control. In oneaspect, the subject is a human.

The invention also provides methods for preventing, treating and/ormanaging a disorder associated with mesothelin-expressing cells (e.g.,mesothelioma), the methods comprising administering to a subject in needa mesothelioma CAR-expressing cell of the invention that binds to themesothelin-expressing cell. In one aspect, the subject is a human.

The invention provides methods for preventing relapse of cancerassociated with mesothelin-expressing cells, the methods comprisingadministering to a subject in need thereof a mesothelin CAR-expressingcell of the invention that binds to the mesothelin-expressing cell. Inanother embodiment, the methods comprise administering to the subject inneed thereof an effective amount of a mesothelin CAR-expressing cell ofthe invention that binds to the mesothelin-expressing cell incombination with an effective amount of another therapy.

In one aspect, the invention pertains to a vector comprising a sequenceencoding a mesothelin CAR operably linked to promoter for expression inmammalian immune effector cells. In one aspect, the invention provides arecombinant immune effector cell expressing the mesothelin CAR for usein treating mesothelin-expressing tumors. In one aspect, the mesothelinCAR-expressing cell of the invention is capable of contacting a tumorcell with at least one mesothelin CAR of the invention expressed on itssurface such that the mesothelin CAR-expressing cell is activated inresponse to the antigen and the CAR-expressing cell targets the cancercell and growth of the cancer is inhibited.

In one aspect, the invention pertains to a method of inhibiting growthof a mesothelin-expressing cancer cell, comprising contacting the tumorcell with a-mesothelin CAR-expressing cell such that the CAR-expressingcell is activated in response to the antigen and targets the cancercell, wherein the growth of the cancer is inhibited. In one aspect, theactivated CART targets and kills the cancer cell.

In one aspect, the invention pertains to a method of treating cancer ina subject. The method comprises administering to the subject amesothelin CAR-expressing cell such that the cancer is treated in thesubject. An example of a cancer that is treatable by the mesothelinCAR-expressing cell of the invention is a cancer associated withexpression of mesothelin. In one aspect, the cancer associated withexpression of mesothelin is selected from mesothelioma, pancreaticcancer, ovarian cancer and lung cancer.

The invention includes a type of cellular therapy where immune effectorcells, e.g., T cells or NK cells, are genetically modified to express achimeric antigen receptor (CAR) and the CAR-expressing cell is infusedto a recipient in need thereof. The infused cell is able to kill tumorcells in the recipient. Unlike antibody therapies, CAR-modified immuneeffector cells are able to replicate in vivo resulting in long-termpersistence that can lead to sustained tumor control. In variousaspects, the cells administered to the patient, or their progeny,persist in the patient for at least four months, five months, sixmonths, seven months, eight months, nine months, ten months, elevenmonths, twelve months, thirteen months, fourteen month, fifteen months,sixteen months, seventeen months, eighteen months, nineteen months,twenty months, twenty-one months, twenty-two months, twenty-threemonths, two years, three years, four years, or five years afteradministration of the cell to the patient.

The invention also includes a type of cellular therapy where immuneeffector cells are modified, e.g., by in vitro transcribed RNA, totransiently express a chimeric antigen receptor (CAR) and theCAR-expressing cell is infused to a recipient in need thereof. Theinfused cell is able to kill cancer cells in the recipient. Thus, invarious aspects, the cells administered to the patient, is present forless than one month, e.g., three weeks, two weeks, one week, afteradministration of the cell to the patient.

Without wishing to be bound by any particular theory, the anti-cancerimmunity response elicited by the CAR-modified immune effector cells maybe an active or a passive immune response, or alternatively may be dueto a direct vs indirect immune response. In one aspect, the CARtransduced T cells exhibit specific proinflammatory cytokine secretionand potent cytolytic activity in response to human cancer cellsexpressing mesothelin, and mediate bystander killing and mediateregression of an established human tumor. For example, antigen-lesstumor cells within a heterogeneous field of mesothelin-expressing tumormay be susceptible to indirect destruction by mesothelin-redirected Tcells that has previously reacted against adjacent antigen-positivecancer cells.

In one aspect, the fully-human scFv bearing CAR-modified immune effectorcells of the invention may be a type of vaccine for ex vivo immunizationand/or in vivo therapy in a mammal. In one aspect, the mammal is ahuman.

With respect to ex vivo immunization, at least one of the followingoccurs in vitro prior to administering the cell into a mammal: i)expansion of the cells, ii) introducing a nucleic acid encoding a CAR tothe cells or iii) cryopreservation of the cells.

Ex vivo procedures are well known in the art and are discussed morefully below. Briefly, cells are isolated from a mammal (e.g., a human)and genetically modified (i.e., transduced or transfected in vitro) witha vector expressing a CAR disclosed herein. The CAR-modified cell can beadministered to a mammalian recipient to provide a therapeutic benefit.The mammalian recipient may be a human and the CAR-modified cell can beautologous with respect to the recipient. Alternatively, the cells canbe allogeneic, syngeneic or xenogeneic with respect to the recipient.

The procedure for ex vivo expansion of hematopoietic stem and progenitorcells is described in U.S. Pat. No. 5,199,942, incorporated herein byreference, can be applied to the cells of the present invention. Othersuitable methods are known in the art therefore the present invention isnot limited to any particular method of ex vivo expansion of the cells.Briefly, ex vivo culture and expansion of T cells comprises: (1)collecting CD34+ hematopoietic stem and progenitor cells from a mammalfrom peripheral blood harvest or bone marrow explants; and (2) expandingsuch cells ex vivo. In addition to the cellular growth factors describedin U.S. Pat. No. 5,199,942, other factors such as flt3-L, IL-1, IL-3 andc-kit ligand, can be used for culturing and expansion of the cells.

In addition to using a cell-based vaccine in terms of ex vivoimmunization, the present invention also provides compositions andmethods for in vivo immunization to elicit an immune response directedagainst an antigen in a patient.

Generally, the cells activated and expanded as described herein may beutilized in the treatment and prevention of diseases that arise inindividuals who are immunocompromised. In particular, the CAR-modifiedimmune effector cells of the invention are used in the treatment ofdiseases, disorders and conditions associated with expression ofmesothelin. In certain aspects, the cells of the invention are used inthe treatment of patients at risk for developing diseases, disorders andconditions associated with expression of mesothelin. Thus, the inventionprovides methods for the treatment or prevention of diseases, disordersand conditions associated with expression of mesothelin comprisingadministering to a subject in need thereof, a therapeutically effectiveamount of the CAR-modified T cells of the invention.

The CAR-modified T cells of the present invention may be administeredeither alone, or as a pharmaceutical composition in combination withdiluents and/or with other components such as IL-2 or other cytokines orcell populations.

The present invention also provides methods for inhibiting theproliferation or reducing a mesothelin-expressing cell population, themethods comprising contacting a population of cells comprising amesothelin-expressing cell with a mesothelin CAR-expressing cell (e.g.,a mesothelin CART also referred to as “CART-MSLN”) of the invention thatbinds to the mesothelin-expressing cell. In a specific aspect, theinvention provides methods for inhibiting the proliferation or reducingthe population of cancer cells expressing mesothelin, the methodscomprising contacting the mesothelin-expressing cancer cell populationwith a mesothelin CAR-expressing cell of the invention that binds to themesothelin-expressing cell. In one aspect, the present inventionprovides methods for inhibiting the proliferation or reducing thepopulation of cancer cells expressing mesothelin, the methods comprisingcontacting the mesothelin-expressing cancer cell population with amesothelin CAR-expressing cell of the invention that binds to themesothelin-expressing cell. In certain aspects, the mesothelinCAR-expressing cell of the invention reduces the quantity, number,amount or percentage of cells and/or cancer cells by at least 25%, atleast 30%, at least 40%, at least 50%, at least 65%, at least 75%, atleast 85%, at least 95%, or at least 99% in a subject with or animalmodel for mesothelioma or another cancer associated withmesothelin-expressing cells relative to a negative control. In oneaspect, the subject is a human.

The present invention also provides methods for preventing, treatingand/or managing a disease associated with mesothelin-expressing cells(e.g., mesothelioma), the methods comprising administering to a subjectin need a mesothelin CAR-expressing cell of the invention that binds tothe mesothelin-expressing cell. In one aspect, the subject is a human.

The present invention provides methods for preventing relapse of cancerassociated with mesothelin-expressing cells, the methods comprisingadministering to a subject in need thereof a mesothelin CAR-expressingcell of the invention that binds to the mesothelin-expressing cell. Inone aspect, the methods comprise administering to the subject in needthereof an effective amount of a mesothelin CAR-expressing cell of theinvention that binds to the mesothelin-expressing cell in combinationwith an effective amount of another therapy.

Combination Therapies

A CAR-expressing cell described herein may be used in combination withother known agents and therapies. Administered “in combination”, as usedherein, means that two (or more) different treatments are delivered tothe subject during the course of the subject's affliction with thedisorder, e.g., the two or more treatments are delivered after thesubject has been diagnosed with the disorder and before the disorder hasbeen cured or eliminated or treatment has ceased for other reasons. Insome embodiments, the delivery of one treatment is still occurring whenthe delivery of the second begins, so that there is overlap in terms ofadministration. This is sometimes referred to herein as “simultaneous”or “concurrent delivery”. In other embodiments, the delivery of onetreatment ends before the delivery of the other treatment begins. Insome embodiments of either case, the treatment is more effective becauseof combined administration. For example, the second treatment is moreeffective, e.g., an equivalent effect is seen with less of the secondtreatment, or the second treatment reduces symptoms to a greater extent,than would be seen if the second treatment were administered in theabsence of the first treatment, or the analogous situation is seen withthe first treatment. In some embodiments, delivery is such that thereduction in a symptom, or other parameter related to the disorder isgreater than what would be observed with one treatment delivered in theabsence of the other. The effect of the two treatments can be partiallyadditive, wholly additive, or greater than additive. The delivery can besuch that an effect of the first treatment delivered is still detectablewhen the second is delivered.

A CAR-expressing cell described herein and the at least one additionaltherapeutic agent can be administered simultaneously, in the same or inseparate compositions, or sequentially. For sequential administration,the CAR-expressing cell described herein can be administered first, andthe additional agent can be administered second, or the order ofadministration can be reversed.

In further aspects, a CAR-expressing cell described herein may be usedin a treatment regimen in combination with surgery, chemotherapy,radiation, immunosuppressive agents, such as cyclosporin, azathioprine,methotrexate, mycophenolate, and FK506, antibodies, or otherimmunoablative agents such as CAMPATH, anti-CD3 antibodies or otherantibody therapies, cytoxin, fludarabine, cyclosporin, FK506, rapamycin,mycophenolic acid, steroids, FR901228, cytokines, and irradiation.peptide vaccine, such as that described in Izumoto et al. 2008 JNeurosurg 108:963-971.

In one embodiment, a CAR-expressing cell described herein can be used incombination with a chemotherapeutic agent. Exemplary chemotherapeuticagents include an anthracycline (e.g., doxorubicin (e.g., liposomaldoxorubicin)). a vinca alkaloid (e.g., vinblastine, vincristine,vindesine, vinorelbine), an alkylating agent (e.g., cyclophosphamide,decarbazine, melphalan, ifosfamide, temozolomide), an immune cellantibody (e.g., alemtuzamab, gemtuzumab, rituximab, tositumomab), anantimetabolite (including, e.g., folic acid antagonists, pyrimidineanalogs, purine analogs and adenosine deaminase inhibitors (e.g.,fludarabine)), an mTOR inhibitor, a TNFR glucocorticoid induced TNFRrelated protein (GITR) agonist, a proteasome inhibitor (e.g.,aclacinomycin A, gliotoxin or bortezomib), an immunomodulator such asthalidomide or a thalidomide derivative (e.g., lenalidomide).

General Chemotherapeutic agents considered for use in combinationtherapies include anastrozole (Arimidex®), bicalutamide (Casodex®),bleomycin sulfate (Blenoxane®), busulfan (Myleran®), busulfan injection(Busulfex®), capecitabine (Xeloda®),N4-pentoxycarbonyl-5-deoxy-5-fluorocytidine, carboplatin (Paraplatin®),carmustine (BiCNU®), chlorambucil (Leukeran®), cisplatin (Platinol®),cladribine (Leustatin®), cyclophosphamide (Cytoxan® or Neosar®),cytarabine, cytosine arabinoside (Cytosar-U®), cytarabine liposomeinjection (DepoCyt®), dacarbazine (DTIC-Dome®), dactinomycin(Actinomycin D, Cosmegan), daunorubicin hydrochloride (Cerubidine®),daunorubicin citrate liposome injection (DaunoXome®), dexamethasone,docetaxel (Taxotere®), doxorubicin hydrochloride (Adriamycin®, Rubex®),etoposide (Vepesid®), fludarabine phosphate (Fludara®), 5-fluorouracil(Adrucil®, Efudex®), flutamide (Eulexin®), tezacitibine, Gemcitabine(difluorodeoxycitidine), hydroxyurea (Hydrea®), Idarubicin (Idamycin®),ifosfamide (IFEX®), irinotecan (Camptosar®), L-asparaginase (ELSPAR®),leucovorin calcium, melphalan (Alkeran®), 6-mercaptopurine(Purinethol®), methotrexate (Folex®), mitoxantrone (Novantrone®),mylotarg, paclitaxel (Taxol®), phoenix (Yttrium90/MX-DTPA), pentostatin,polifeprosan 20 with carmustine implant (Gliadel®), tamoxifen citrate(Nolvadex®), teniposide (Vumon®), 6-thioguanine, thiotepa, tirapazamine(Tirazone®), topotecan hydrochloride for injection (Hycamptin®),vinblastine (Velban®), vincristine (Oncovin®), and vinorelbine(Navelbine®).

Exemplary alkylating agents include, without limitation, nitrogenmustards, ethylenimine derivatives, alkyl sulfonates, nitrosoureas andtriazenes): uracil mustard (Aminouracil Mustard®, Chlorethaminacil®,Demethyldopan®, Desmethyldopan®, Haemanthamine®, Nordopan®, Uracilnitrogen Mustard®, Uracillost®, Uracilmostaza®, Uramustin®,Uramustine®), chlormethine (Mustargen®), cyclophosphamide (Cytoxan®,Neosar®, Clafen®, Endoxan®, Procytox®, Revimmune™), ifosfamide(Mitoxana®), melphalan (Alkeran®), Chlorambucil (Leukeran®), pipobroman(Amedel®, Vercyte®), triethylenemelamine (Hemel®, Hexalen®, Hexastat®),triethylenethiophosphoramine, Temozolomide (Temodar®), thiotepa(Thioplex®), busulfan (Busilvex®, Myleran®), carmustine (BiCNU®),lomustine (CeeNU®), streptozocin (Zanosar®), and Dacarbazine(DTIC-Dome®). Additional exemplary alkylating agents include, withoutlimitation, Oxaliplatin (Eloxatin®); Temozolomide (Temodar® andTemodal®); Dactinomycin (also known as actinomycin-D, Cosmegen®);Melphalan (also known as L-PAM, L-sarcolysin, and phenylalanine mustard,Alkeran®); Altretamine (also known as hexamethylmelamine (HMM),Hexalen®); Carmustine (BiCNU®); Bendamustine (Treanda®); Busulfan(Busulfex® and Myleran®); Carboplatin (Paraplatin®); Lomustine (alsoknown as CCNU, CeeNU®); Cisplatin (also known as CDDP, Platinol® andPlatinol®-AQ); Chlorambucil (Leukeran®); Cyclophosphamide (Cytoxan® andNeosar®); Dacarbazine (also known as DTIC, DIC and imidazolecarboxamide, DTIC-Dome®); Altretamine (also known as hexamethylmelamine(HMM), Hexalen®); Ifosfamide (Ifex®); Prednumustine; Procarbazine(Matulane®); Mechlorethamine (also known as nitrogen mustard, mustineand mechloroethamine hydrochloride, Mustargen®); Streptozocin(Zanosar®); Thiotepa (also known as thiophosphoamide, TESPA and TSPA,Thioplex®); Cyclophosphamide (Endoxan®, Cytoxan®, Neosar®, Procytox®,Revimmune®); and Bendamustine HCl (Treandag).

Exemplary mTOR inhibitors include, e.g., temsirolimus; ridaforolimus(formally known as deferolimus, (1R,2R,4S)-4-[(2R)-2[(1R,9S,12S,15R,16E,18R,19R,21R, 23S,24E,26E,28Z,30S,32S,35R)-1,18-dihydroxy-19,30-dimethoxy-15,17,21,23,29,35-hexamethyl-2,3,10,14,20-pentaoxo-11,36-dioxa-4-azatricyclo[30.3.1.04,9]hexatriaconta-16,24,26,28-tetraen-12-yl]propyl]-2-methoxycyclohexyldimethylphosphinate, also known as AP23573 and MK8669, and described inPCT Publication No. WO 03/064383); everolimus (Afinitor® or RAD001);rapamycin (AY22989, Sirolimus®); simapimod (CAS 164301-51-3);emsirolimus, (5-{2,4-Bis[(3S)-3-methylmorpholin-4-yl]pyrido[2,3-d]pyrimidin-7-yl}-2-methoxyphenyl)methanol(AZD8055);2-Amino-8-[trans-4-(2-hydroxyethoxy)cyclohexyl]-6-(6-methoxy-3-pyridinyl)-4-methyl-pyrido[2,3-d]pyrimidin-7(8H)-one(PF04691502, CAS 1013101-36-4); andN2-[1,4-dioxo-4-[[4-(4-oxo-8-phenyl-4H-1-benzopyran-2-yl)morpholinium-4-yl]methoxy]butyl]-L-arginylglycyl-L-α-aspartylL-serine-(SEQID NO: 402), inner salt (SF1126, CAS 936487-67-1), and XL765. Exemplaryimmunomodulators include, e.g., afutuzumab (available from Roche®);pegfilgrastim (Neulasta®); lenalidomide (CC-5013, Revlimid®);thalidomide (Thalomid®), actimid (CC4047); and IRX-2 (mixture of humancytokines including interleukin 1, interleukin 2, and interferon γ, CAS951209-71-5, available from IRX Therapeutics).

Exemplary anthracyclines include, e.g., doxorubicin (Adriamycin® andRubex®); bleomycin (Lenoxane®); daunorubicin (dauorubicin hydrochloride,daunomycin, and rubidomycin hydrochloride, Cerubidine®); daunorubicinliposomal (daunorubicin citrate liposome, DaunoXome®); mitoxantrone(DHAD, Novantrone®); epirubicin (Ellence™); idarubicin (Idamycin®,Idamycin PFS®); mitomycin C (Mutamycin®); geldanamycin; herbimycin;ravidomycin; and desacetylravidomycin.

Exemplary vinca alkaloids include, e.g., vinorelbine tartrate(Navelbine®), Vincristine (Oncovin®), and Vindesine (Eldisine®));vinblastine (also known as vinblastine sulfate, vincaleukoblastine andVLB, Alkaban-AQ® and Velban®); and vinorelbine (Navelbine®).

Exemplary proteosome inhibitors include bortezomib (Velcade®);carfilzomib (PX-171-007,(S)-4-Methyl-N—((S)-1-(((S)-4-methyl-1-((R)-2-methyloxiran-2-yl)-1-oxopentan-2-yl)amino)-1-oxo-3-phenylpropan-2-yl)-2-((S)-2-(2-morpholinoacetamido)-4-phenylbutanamido)-pentanamide);marizomib (NPI-0052); ixazomib citrate (MLN-9708); delanzomib(CEP-18770); andO-Methyl-N-[(2-methyl-5-thiazolyl)carbonyl]-L-seryl-O-methyl-N-[(1S)-2-[(2R)-2-methyl-2-oxiranyl]-2-oxo-1-(phenylmethyl)ethyl]-L-serinamide(ONX-0912).

In one embodiment, a CAR expressing cell described herein isadministered to a subject in combination with a molecule targeting GITRand/or modulating GITR functions, such as a GITR agonist and/or a GITRantibody that depletes regulatory T cells (Tregs). In one embodiment,the GITR binding molecules and/or molecules modulating GITR functions(e.g., GITR agonist and/or Treg depleting GITR antibodies) areadministered prior to the CAR-expressing cell. For example, in oneembodiment, the GITR agonist can be administered prior to apheresis ofthe cells. Exemplary GITR agonists include, e.g., GITR fusion proteinsand anti-GITR antibodies (e.g., bivalent anti-GITR antibodies) such as,e.g., a GITR fusion protein described in U.S. Pat. No. 6,111,090,European Patent No.: 090505B1, U.S. Pat. No. 8,586,023, PCT PublicationNos.: WO 2010/003118 and 2011/090754, or an anti-GITR antibodydescribed, e.g., in U.S. Pat. No. 7,025,962, European Patent No.:1947183B1, U.S. Pat. Nos. 7,812,135, 8,388,967, 8,591,886, EuropeanPatent No.: EP 1866339, PCT Publication No.: WO 2011/028683, PCTPublication No.: WO 2013/039954, PCT Publication No.: WO2005/007190, PCTPublication No.: WO 2007/133822, PCT Publication No.: WO2005/055808, PCTPublication No.: WO 99/40196, PCT Publication No.: WO 2001/03720, PCTPublication No.: WO99/20758, PCT Publication No.: WO2006/083289, PCTPublication No.: WO 2005/115451, U.S. Pat. No. 7,618,632, and PCTPublication No.: WO 2011/051726.

In one embodiment, a CAR expressing cell described herein isadministered to a subject in combination with an mTOR inhibitor, e.g.,an mTOR inhibitor described herein, e.g., a rapalog such as everolimus.In one embodiment, the mTOR inhibitor is administered prior to theCAR-expressing cell. For example, in one embodiment, the mTOR inhibitorcan be administered prior to apheresis of the cells.

In one embodiment, a CAR expressing cell described herein isadministered to a subject in combination with a GITR agonist, e.g., aGITR agonist described herein. In one embodiment, the GITR agonist isadministered prior to the CAR-expressing cell. For example, in oneembodiment, the GITR agonist can be administered prior to apheresis ofthe cells.

In one embodiment, a CAR expressing cell described herein isadministered to a subject in combination with a protein tyrosinephosphatase inhibitor, e.g., a protein tyrosine phosphatase inhibitordescribed herein. In one embodiment, the protein tyrosine phosphataseinhibitor is an SHP-1 inhibitor, e.g., an SHP-1 inhibitor describedherein, such as, e.g., sodium stibogluconate. In one embodiment, theprotein tyrosine phosphatase inhibitor is an SHP-2 inhibitor, e.g., anSHP-2 inhibitor described herein.

In one embodiment, a CAR-expressing cell described herein can be used incombination with a kinase inhibitor. In one embodiment, the kinaseinhibitor is a CDK4 inhibitor, e.g., a CDK4 inhibitor described herein,e.g., a CDK4/6 inhibitor, such as, e.g.,6-Acetyl-8-cyclopentyl-5-methyl-2-(5-piperazin-1-yl-pyridin-2-ylamino)-8H-pyrido[2,3-d]pyrimidin-7-one,hydrochloride (also referred to as palbociclib or PD0332991). In oneembodiment, the kinase inhibitor is a BTK inhibitor, e.g., a BTKinhibitor described herein, such as, e.g., ibrutinib. In one embodiment,the kinase inhibitor is an mTOR inhibitor, e.g., an mTOR inhibitordescribed herein, such as, e.g., rapamycin, a rapamycin analog, OSI-027.The mTOR inhibitor can be, e.g., an mTORC1 inhibitor and/or an mTORC2inhibitor, e.g., an mTORC1 inhibitor and/or mTORC2 inhibitor describedherein. In one embodiment, the kinase inhibitor is a MNK inhibitor,e.g., a MNK inhibitor described herein, such as, e.g.,4-amino-5-(4-fluoroanilino)-pyrazolo [3,4-d] pyrimidine. The MNKinhibitor can be, e.g., a MNKla, MNK1b, MNK2a and/or MNK2b inhibitor. Inone embodiment, the kinase inhibitor is a dual PI3K/mTOR inhibitordescribed herein, such as, e.g., PF-04695102. In one embodiment, thekinase inhibitor is a DGK inhibitor, e.g., a DGK inhibitor describedherein, such as, e.g., DGKinh1 (D5919) or DGKinh2 (D5794).

In one embodiment, the kinase inhibitor is a CDK4 inhibitor selectedfrom aloisine A; flavopiridol or HMR-1275,2-(2-chlorophenyl)-5,7-dihydroxy-8-[(3S,4R)-3-hydroxy-1-methyl-4-piperidinyl]-4-chromenone;crizotinib (PF-02341066;2-(2-Chlorophenyl)-5,7-dihydroxy-8-[(2R,3S)-2-(hydroxymethyl)-1-methyl-3-pyrrolidinyl]-4H-1-benzopyran-4-one,hydrochloride (P276-00);1-methyl-5[[2-[5-(trifluoromethyl)-1H-imidazol-2-yl]-4-pyridinyl]oxy]-N-[4-(trifluoromethyl)phenyl]-1H-benzimidazol-2-amine(RAF265); indisulam (E7070); roscovitine (CYC202); palbociclib(PD0332991); dinaciclib (SCH727965);N-[5-[[(5-tert-butyloxazol-2-yl)methyl]thio]thiazol-2-yl]piperidine-4-carboxamide(BMS 387032);4-[[9-chloro-7-(2,6-difluorophenyl)-5H-pyrimido[5,4-d][2]benzazepin-2-yl]amino]-benzoicacid (MLN8054);5-[3-(4,6-difluoro-1H-benzimidazol-2-yl)-1H-indazol-5-yl]-N-ethyl-4-methyl-3-pyridinemethanamine(AG-024322); 4-(2,6-dichlorobenzoylamino)-1H-pyrazole-3-carboxylic acidN-(piperidin-4-yl)amide (AT7519);4-[2-methyl-1-(1-methylethyl)-1H-imidazol-5-yl]-N-[4-(methylsulfonyl)phenyl]-2-pyrimidinamine(AZD5438); and XL281 (BMS908662).

In one embodiment, the kinase inhibitor is a CDK4 inhibitor, e.g.,palbociclib (PD0332991), and the palbociclib is administered at a doseof about 50 mg, 60 mg, 70 mg, 75 mg, 80 mg, 90 mg, 100 mg, 105 mg, 110mg, 115 mg, 120 mg, 125 mg, 130 mg, 135 mg (e.g., 75 mg, 100 mg or 125mg) daily for a period of time, e.g., daily for 14-21 days of a 28 daycycle, or daily for 7-12 days of a 21 day cycle. In one embodiment, 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more cycles of palbociclib areadministered.

In one embodiment, the kinase inhibitor is a BTK inhibitor selected fromibrutinib (PCI-32765); GDC-0834; RN-486; CGI-560; CGI-1764; HM-71224;CC-292; ONO-4059; CNX-774; and LFM-A13. In a preferred embodiment, theBTK inhibitor does not reduce or inhibit the kinase activity ofinterleukin-2-inducible kinase (ITK), and is selected from GDC-0834;RN-486; CGI-560; CGI-1764; HM-71224; CC-292; ONO-4059; CNX-774; andLFM-A13.

In one embodiment, the kinase inhibitor is a BTK inhibitor, e.g.,ibrutinib (PCI-32765), and the ibrutinib is administered at a dose ofabout 250 mg, 300 mg, 350 mg, 400 mg, 420 mg, 440 mg, 460 mg, 480 mg,500 mg, 520 mg, 540 mg, 560 mg, 580 mg, 600 mg (e.g., 250 mg, 420 mg or560 mg) daily for a period of time, e.g., daily for 21 day cycle cycle,or daily for 28 day cycle. In one embodiment, 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12 or more cycles of ibrutinib are administered.

In one embodiment, the kinase inhibitor is an mTOR inhibitor selectedfrom temsirolimus; ridaforolimus (1R,2R,4S)-4-[(2R)-2[(1R,9S,12S,15R,16E,18R,19R,21R,23S,24E,26E,28Z,30S,32S,35R)-1,18-dihydroxy-19,30-dimethoxy-15,17,21,23,29,35-hexamethyl-2,3,10,14,20-pentaoxo-11,36-dioxa-4-azatricyclo[30.3.1.0^(4,9)]hexatriaconta-16,24,26,28-tetraen-12-yl]propyl]-2-methoxycyclohexyldimethylphosphinate, also known as AP23573 and MK8669; everolimus(RAD001); rapamycin (AY22989); simapimod;(5-{2,4-bis[(3S)-3-methylmorpholin-4-yl]pyrido[2,3-d]pyrimidin-7-yl}-2-methoxyphenyl)methanol(AZD8055);2-amino-8-[trans-4-(2-hydroxyethoxy)cyclohexyl]-6-(6-methoxy-3-pyridinyl)-4-methyl-pyrido[2,3-d]pyrimidin-7(8H)-one(PF04691502); andN²-[1,4-dioxo-4-[[4-(4-oxo-8-phenyl-4H-1-benzopyran-2-yl)morpholinium-4-yl]methoxy]buty]R-arginylglycyl-L-α-aspartylL-serine-(SEQID NO: 272), inner salt (SF1126); and XL765.

In one embodiment, the kinase inhibitor is an mTOR inhibitor, e.g.,rapamycin, and the rapamycin is administered at a dose of about 3 mg, 4mg, 5 mg, 6 mg, 7 mg, 8 mg, 9 mg, 10 mg (e.g., 6 mg) daily for a periodof time, e.g., daily for 21 day cycle cycle, or daily for 28 day cycle.In one embodiment, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more cyclesof rapamycin are administered. In one embodiment, the kinase inhibitoris an mTOR inhibitor, e.g., everolimus and the everolimus isadministered at a dose of about 2 mg, 2.5 mg, 3 mg, 4 mg, 5 mg, 6 mg, 7mg, 8 mg, 9 mg, 10 mg, 11 mg, 12 mg, 13 mg, 14 mg, 15 mg (e.g., 10 mg)daily for a period of time, e.g., daily for 28 day cycle. In oneembodiment, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more cycles ofeverolimus are administered.

In one embodiment, the kinase inhibitor is an MNK inhibitor selectedfrom CGP052088; 4-amino-3-(p-fluorophenylamino)-pyrazolo [3,4-d]pyrimidine (CGP57380); cercosporamide; ETC-1780445-2; and4-amino-5-(4-fluoroanilino)-pyrazolo [3,4-d] pyrimidine.

In one embodiment, the kinase inhibitor is a dual phosphatidylinositol3-kinase (PI3K) and mTOR inhibitor selected from2-Amino-8-[trans-4-(2-hydroxyethoxy)cyclohexyl]-6-(6-methoxy-3-pyridinyl)-4-methyl-pyrido[2,3-d]pyrimidin-7(8H)-one(PF-04691502);N-[4-[[4-(Dimethylamino)-1-piperidinyl]carbonyl]phenyl]-N-[4-(4,6-di-4-morpholinyl-1,3,5-triazin-2-yl)phenyl]urea(PF-05212384, PKI-587);2-Methyl-2-{4-[3-methyl-2-oxo-8-(quinolin-3-yl)-2,3-dihydro-1H-imidazo[4,5-c]quinolin-1-yl]phenyl}propanenitrile(BEZ-235); apitolisib (GDC-0980, RG7422);2,4-Difluoro-N-{2-(methyloxy)-5-[4-(4-pyridazinyl)-6-quinolinyl]-3-pyridinyl}benzenesulfonamide(GSK2126458);8-(6-methoxypyridin-3-yl)-3-methyl-1-(4-(piperazin-1-yl)-3-(trifluoromethyl)phenyl)-1H-imidazo[4,5-c]quinolin-2(3H)-oneMaleic acid (NVP-BGT226);3-[4-(4-Morpholinylpyrido[3′,2′:4,5]furo[3,2-d]pyrimidin-2-yl]phenol(PI-103);5-(9-isopropyl-8-methyl-2-morpholino-9H-purin-6-yl)pyrimidin-2-amine(VS-5584, SB2343); andN-[2-[(3,5-Dimethoxyphenyl)amino]quinoxalin-3-yl]-4-[(4-methyl-3-methoxyphenyl)carbonyl]aminophenylsulfonamide(XL765).

Drugs that inhibit either the calcium dependent phosphatase calcineurin(cyclosporine and FK506) or inhibit the p70S6 kinase that is importantfor growth factor induced signaling (rapamycin). (Liu et al., Cell66:807-815, 1991; Henderson et al., Immun. 73:316-321, 1991; Bierer etal., Curr. Opin. Immun. 5:763-773, 1993) can also be used. In a furtheraspect, the cell compositions of the present invention may beadministered to a patient in conjunction with (e.g., before,simultaneously or following) bone marrow transplantation, T cellablative therapy using chemotherapy agents such as, fludarabine,external-beam radiation therapy (XRT), cyclophosphamide, and/orantibodies such as OKT3 or CAMPATH. In one aspect, the cell compositionsof the present invention are administered following B-cell ablativetherapy such as agents that react with CD20, e.g., Rituxan. For example,in one embodiment, subjects may undergo standard treatment with highdose chemotherapy followed by peripheral blood stem celltransplantation. In certain embodiments, following the transplant,subjects receive an infusion of the expanded immune cells of the presentinvention. In an additional embodiment, expanded cells are administeredbefore or following surgery.

In one embodiment, the subject can be administered an agent whichreduces or ameliorates a side effect associated with the administrationof a CAR-expressing cell. Side effects associated with theadministration of a CAR-expressing cell include, but are not limited toCRS, and hemophagocytic lymphohistiocytosis (HLH), also termedMacrophage Activation Syndrome (MAS). Symptoms of CRS include highfevers, nausea, transient hypotension, hypoxia, and the like. CRS mayinclude clinical constitutional signs and symptoms such as fever,fatigue, anorexia, myalgias, arthalgias, nausea, vomiting, and headache.CRS may include clinical skin signs and symptoms such as rash. CRS mayinclude clinical gastrointestinal signs and symsptoms such as nausea,vomiting and diarrhea. CRS may include clinical respiratory signs andsymptoms such as tachypnea and hypoxemia. CRS may include clinicalcardiovascular signs and symptoms such as tachycardia, widened pulsepressure, hypotension, increased cardac output (early) and potentiallydiminished cardiac output (late). CRS may include clinical coagulationsigns and symptoms such as elevated d-dimer, hypofibrinogenemia with orwithout bleeding. CRS may include clinical renal signs and symptoms suchas azotemia. CRS may include clinical hepatic signs and symptoms such astransaminitis and hyperbilirubinemia. CRS may include clinicalneurologic signs and symptoms such as headache, mental status changes,confusion, delirium, word finding difficulty or frank aphasia,hallucinations, tremor, dymetria, altered gait, and seizures.

Accordingly, the methods described herein can comprise administering aCAR-expressing cell described herein to a subject and furtheradministering one or more agents to manage elevated levels of a solublefactor resulting from treatment with a CAR-expressing cell. In oneembodiment, the soluble factor elevated in the subject is one or more ofIFN-γ, TNFα, IL-2 and IL-6. In an embodiment, the factor elevated in thesubject is one or more of IL-1, GM-CSF, IL-10, IL-8, IL-5 andfraktalkine. Therefore, an agent administered to treat this side effectcan be an agent that neutralizes one or more of these soluble factors.In one embodiment, the agent that neutralizes one or more of thesesoluble forms is an antibody or antigen binding fragment thereof.Examples of such agents include, but are not limited to a steroid (e.g.,corticosteroid), an inhibitor of TNFα, and an inhibitor of IL-6. Anexample of a TNFα inhibitor is an anti-TNFα antibody molecule such as,infliximab, adalimumab, certolizumab pegol, and golimumab. Anotherexample of a TNFα inhibitor is a fusion protein such as entanercept.Small molecule inhibitor of TNFα include, but are not limited to,xanthine derivatives (e.g. pentoxifylline) and bupropion. An example ofan IL-6 inhibitor is an anti-IL-6 antibody molecule or an anti-IL-6receptor antibody molecule such as tocilizumab (toc), sarilumab,elsilimomab, CNTO 328, ALD518/BMS-945429, CNTO 136, CPSI-2364, CDP6038,VX30, ARGX-109, FE301, and FM101. In one embodiment, the anti-IL-6antibody molecule is tocilizumab. An example of an IL-1R based inhibitoris anakinra.

In some embodiment, the subject is administered a corticosteroid, suchas, e.g., methylprednisolone, hydrocortisone, among others.

In some embodiments, the subject is administered a vasopressor, such as,e.g., norepinephrine, dopamine, phenylephrine, epinephrine, vasopressin,or a combination thereof.

In an embodiment, the subject can be administered an antipyretic agent.In an embodiment, the subject can be administered an analgesic agent.

In one embodiment, the subject can be administered an agent whichenhances the activity or fitness of a CAR-expressing cell. For example,in one embodiment, the agent can be an agent which inhibits a moleculethat modulates or regulates, e.g., inhibits, T cell function. In someembodiments, the molecule that modulates or regulates T cell function isan inhibitory molecule. Inhibitory molecules, e.g., Programmed Death 1(PD1), can, in some embodiments, decrease the ability of aCAR-expressing cell to mount an immune effector response. Examples ofinhibitory molecules include PD1, PD-L1, CTLA4, TIM3, CEACAM (e.g.,CEACAM-1, CEACAM-3 and/or CEACAM-5), LAG3, VISTA, BTLA, TIGIT, LAIR1,CD160, 2B4 and TGFR beta. Inhibition of a molecule that modulates orregulates, e.g., inhibits, T cell function, e.g., by inhibition at theDNA, RNA or protein level, can optimize a CAR-expressing cellperformance. In embodiments, an agent, e.g., an inhibitory nucleic acid,e.g., a dsRNA, e.g., an siRNA or shRNA; or e.g., an inhibitory proteinor system, e.g., a clustered regularly interspaced short palindromicrepeats (CRISPR), a transcription-activator like effector nuclease(TALEN), or a zinc finger endonuclease (ZFN), e.g., as described herein,can be used to inhibit expression of a molecule that modulates orregulates, e.g., inhibits, T-cell function in the CAR-expressing cell.In an embodiment the agent is an shRNA. In an embodiment, the agent thatmodulates or regulates, e.g., inhibits, T-cell function is inhibitedwithin a CAR-expressing cell. In these embodiments, a dsRNA moleculethat inhibits expression of a molecule that modulates or regulates,e.g., inhibits, T-cell function is linked to the nucleic acid thatencodes a component, e.g., all of the components, of the CAR. In anembodiment, a nucleic acid molecule that encodes a dsRNA molecule thatinhibits expression of the molecule that modulates or regulates, e.g.,inhibits, T-cell function is operably linked to a promoter, e.g., a H1-or a U6-derived promoter such that the dsRNA molecule that inhibitsexpression of the molecule that modulates or regulates, e.g., inhibits,T-cell function is expressed, e.g., is expressed within a CAR-expressingcell. See e.g., Tiscornia G., “Development of Lentiviral VectorsExpressing siRNA,” Chapter 3, in Gene Transfer: Delivery and Expressionof DNA and RNA (eds. Friedmann and Rossi). Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y., USA, 2007; Brummelkamp T R, et al.(2002) Science 296: 550-553; Miyagishi M, et al. (2002) Nat. Biotechnol.19: 497-500. In an embodiment the nucleic acid molecule that encodes adsRNA molecule that inhibits expression of the molecule that modulatesor regulates, e.g., inhibits, T-cell function is present on the samevector, e.g., a lentiviral vector, that comprises a nucleic acidmolecule that encodes a component, e.g., all of the components, of theCAR. In such an embodiment, the nucleic acid molecule that encodes adsRNA molecule that inhibits expression of the molecule that modulatesor regulates, e.g., inhibits, T-cell function is located on the vector,e.g., the lentiviral vector, 5′- or 3′- to the nucleic acid that encodesa component, e.g., all of the components, of the CAR. The nucleic acidmolecule that encodes a dsRNA molecule that inhibits expression of themolecule that modulates or regulates, e.g., inhibits, T-cell functioncan be transcribed in the same or different direction as the nucleicacid that encodes a component, e.g., all of the components, of the CAR.In an embodiment the nucleic acid molecule that encodes a dsRNA moleculethat inhibits expression of the molecule that modulates or regulates,e.g., inhibits, T-cell function is present on a vector other than thevector that comprises a nucleic acid molecule that encodes a component,e.g., all of the components, of the CAR. In an embodiment, the nucleicacid molecule that encodes a dsRNA molecule that inhibits expression ofthe molecule that modulates or regulates, e.g., inhibits, T-cellfunction it transiently expressed within a CAR-expressing cell. In anembodiment, the nucleic acid molecule that encodes a dsRNA molecule thatinhibits expression of the molecule that modulates or regulates, e.g.,inhibits, T-cell function is stably integrated into the genome of aCAR-expressing cell. FIG. 47 depicts examples of vectors for expressinga component, e.g., all of the components, of the CAR with a dsRNAmolecule that inhibits expression of the molecule that modulates orregulates, e.g., inhibits, T-cell function.

Examples of dsRNA molecules useful for inhibiting expression of amolecule that modulates or regulates, e.g., inhibits, T-cell function,wherein the molecule that modulates or regulates, e.g., inhibits, T-cellfunction is PD-1 are provided below.

Provided in Table 16 below are the names of PDCD1 (PD1) RNAi agents(derived from their position in the mouse PDCD1 gene sequenceNM_008798.2), along with the SEQ ID NOs: 280-327 representing the DNAsequence. Both sense (S) and antisense (AS) sequences are presented as19mer and 21mer sequences are in this table. Also note that the position(PoS, e.g., 176) is derived from the position number in the mouse PDCD1gene sequence NM_008798.2. SEQ ID NOs are indicated in groups of 12 thatcorrespond with “sense 19” SEQ ID NOs: 280-291; “sense 21” SEQ ID NOs:292-303; “asense 21” SEQ ID NOs: 304-315; “asense 19” SEQ ID NOs:316-327.

TABLE 16 Mouse PDCD1 (PD1) shRNA sequences Position on TargetNM_008798.2 region Sense19 Sense21 Asense21 Asense19 176 CDS GGAGGTCCCTCCTGGAGGTCCC TAGAAGGTGAG TAGAAGGTGAG ACCTTCTA TCACCTTCTA GGACCTCCAGGGACCTCC (SEQ ID NO: (SEQ ID NO: (SEQ ID NO: (SEQ ID NO: 280) 292) 304)316) 260 CDS CGGAGGATCTT GTCGGAGGATC TTCAGCATAAG TTCAGCATAAG ATGCTGAATTATGCTGAA ATCCTCCGAC ATCCTCCG (SEQ ID NO: (SEQ ID NO: (SEQ ID NO:(SEQ ID NO: 281) 293) 305) 317) 359 CDS CCCGCTTCCAG TGCCCGCTTCCTGTATGATCTG TGTATGATCTG ATCATACA AGATCATACA GAAGCGGGCA GAAGCGGG(SEQ ID NO: (SEQ ID NO: (SEQ ID NO: (SEQ ID NO: 282) 294) 306) 318) 528CDS GGAGACCTCAA CTGGAGACCTC ATATCTTGTTG ATATCTTGTTG CAAGATAT AACAAGATATAGGTCTCCAG AGGTCTCC (SEQ ID NO: (SEQ ID NO: (SEQ ID NO: (SEQ ID NO: 283)295) 307) 319) 581 CDS AAGGCATGGTC TCAAGGCATGG ATACCAATGAC ATACCAATGACATTGGTAT TCATTGGTAT CATGCCTTGA CATGCCTT (SEQ ID NO: (SEQ ID NO:(SEQ ID NO: (SEQ ID NO: 284) 296) 308) 320) 584 CDS GCATGGTCATTAGGCATGGTCA ATGATACCAAT ATGATACCAAT GGTATCAT TTGGTATCAT GACCATGCCTGACCATGC (SEQ ID NO: (SEQ ID NO: (SEQ ID NO: (SEQ ID NO: 285) 297) 309)321) 588 CDS GGTCATTGGTA ATGGTCATTGG ATGGTCATTGG ATGGTCATTGG TCATGAGTTATCATGAGT TATCATGAGT TATCATGA (SEQ ID NO: (SEQ ID NO: (SEQ ID NO:(SEQ ID NO: 286) 298) 310) 322) 609 CDS CCTAGTGGGTA GCCCTAGTGGGCCCTAGTGG GCCCTAGTGG TCCCTGTA GTATCCCTGTA GTATCCCTGTA GTATCCCTG(SEQ ID NO: (SEQ ID NO: (SEQ ID NO: (SEQ ID NO: 287) 299) 311) 323) 919CDS GAGGATGGACA ATGAGGATGGA ATGAGGATGGA ATGAGGATGGA TTGTTCTT CATTGTTCTTCATTGTTCTT CATTGTTC (SEQ ID NO: (SEQ ID NO: (SEQ ID NO: (SEQ ID NO: 288)300) 312) 324) 1021 3′UTR GCATGCAGGCT GAGCATGCAG GAGCATGCAG GAGCATGCAGACAGTTCA GCTACAGTTCA GCTACAGTTCA GCTACAGTT (SEQ ID NO: (SEQ ID NO:(SEQ ID NO: (SEQ ID NO: 289) 301) 313) 325) 1097 3′UTR CCAGCACATGCTTCCAGCACAT TTCCAGCACAT TTCCAGCACAT ACTGTTGA GCACTGTTGA GCACTGTTGAGCACTGTT (SEQ ID NO: (SEQ ID NO: (SEQ ID NO: (SEQ ID NO: 290) 302) 314)326) 1101 3′UTR CACATGCACTG AGCACATGCAC AGCACATGCAC AGCACATGCAC TTGAGTGATGTTGAGTGA TGTTGAGTGA TGTTGAGT (SEQ ID NO: (SEQ ID NO: (SEQ ID NO:(SEQ ID NO: 291) 303) 315) 327)

Provided in Table 17 below are the names of PDCD1 (PD1) RNAi agents(derived from their position in the human PDCD1 gene sequence, alongwith the SEQ ID NOs. 323-370 representing the DNA sequence. Both sense(S) and antisense (AS) sequences are presented as 19mer and 21mersequences. SEQ ID NOs are indicated in groups of 12 that correspond with“sense 19” SEQ ID NOs: 328-339; “sense 21” SEQ ID NOs: 340-351; “asense21” SEQ ID NOs: 352-363; “asense 19” SEQ ID NOs: 364-375.

TABLE 17 Human PDCD1 (PD1) shRNA sequences Position on TargetNM_005018.2 region Sense19 Asense19 Sense21 Asense21 145 CDS GGCCAGGATGTCTAAGAACCA GCGGCCAGGA TCTAAGAACCA GTTCTTAGA TCCTGGCC TGGTTCTTAGATCCTGGCCGC (SEQ ID NO: (SEQ ID NO: (SEQ ID NO: (SEQ ID NO: 328) 340)352) 364) 271 CDS GCTTCGTGCTA TACCAGTTTAG GAGCTTCGTGC TACCAGTTTAGAACTGGTA CACGAAGC TAAACTGGTA CACGAAGCTC (SEQ ID NO: (SEQ ID NO:(SEQ ID NO: (SEQ ID NO: 329) 341) 353) 365) 393 CDS GGGCGTGACTTTCATGTGGAAG ACGGGCGTGA TCATGTGGAAG CCACATGA TCACGCCC CTTCCACATGATCACGCCCGT (SEQ ID NO: (SEQ ID NO: (SEQ ID NO: (SEQ ID NO: 330) 342)354) 366) 1497 3′UTR CAGGCCTAGAG TGAAACTTCTC TGCAGGCCTAG TGAAACTTCTCAAGTTTCA TAGGCCTG AGAAGTTTCA TAGGCCTGCA (SEQ ID NO: (SEQ ID NO:(SEQ ID NO: (SEQ ID NO: 331) 343) 355) 367) 1863 3′UTR CTTGGAACCCATTCAGGAATGG TCCTTGGAACC TTCAGGAATGG TTCCTGAA GTTCCAAG CATTCCTGAAGTTCCAAGGA (SEQ ID NO: (SEQ ID NO: (SEQ ID NO: (SEQ ID NO: 332) 344)356) 368) 1866 3′UTR GGAACCCATTC AATTTCAGGAA TTGGAACCCAT AATTTCAGGAACTGAAATT TGGGTTCC TCCTGAAATT TGGGTTCCAA (SEQ ID NO: (SEQ ID NO:(SEQ ID NO: (SEQ ID NO: 333) 345) 357) 369) 1867 3′UTR GAACCCATTCCTAATTTCAGGA TGGAACCCATT TAATTTCAGGA TGAAATTA ATGGGTTC CCTGAAATTAATGGGTTCCA (SEQ ID NO: (SEQ ID NO: (SEQ ID NO: (SEQ ID NO: 334) 346)358) 370) 1868 3′UTR AACCCATTCCT ATAATTTCAGG GGAACCCATTC ATAATTTCAGGGAAATTAT AATGGGTT CTGAAATTAT AATGGGTTCC (SEQ ID NO: (SEQ ID NO:(SEQ ID NO: (SEQ ID NO: 371) 335) 347) 359) 1869 3′UTR ACCCATTCCTGAATAATTTCAG GAACCCATTCC AATAATTTCAG AAATTATT GAATGGGT TGAAATTATTGAATGGGTTC (SEQ ID NO: (SEQ ID NO: (SEQ ID NO: (SEQ ID NO: 336) 348)360) 372) 1870 3′UTR CCCATTCCTGA AAATAATTTCA AACCCATTCCT AAATAATTTCAAATTATTT GGAATGGG GAAATTATTT GGAATGGGTT (SEQ ID NO: (SEQ ID NO:(SEQ ID NO: (SEQ ID NO: 337) 349) 361) 373) 2079 3′UTR CTGTGGTTCTATAATATAATAGA CCCTGTGGTTC TAATATAATAGA TTATATTA ACCACAG TATTATATTAACCACAGGG (SEQ ID NO: (SEQ ID NO: 350 (SEQ ID NO: (SEQ ID NO: 338) 362)374) 2109 3′UTR AAATATGAGAG TTAGCATGCTC TTAAATATGAG TTAGCATGCTC CATGCTAATCATATTT AGCATGCTAA TCATATTTAA (SEQ ID NO: (SEQ ID NO: (SEQ ID NO:(SEQ ID NO: 339) 351) 363) 375)

In one embodiment, the inhibitor of an inhibitory signal can be, e.g.,an antibody or antibody fragment that binds to an inhibitory molecule.For example, the agent can be an antibody or antibody fragment thatbinds to PD1, PD-L1, PD-L2 or CTLA4 (e.g., ipilimumab (also referred toas MDX-010 and MDX-101, and marketed as Yervoy®; Bristol-Myers Squibb;Tremelimumab (IgG2 monoclonal antibody available from Pfizer, formerlyknown as ticilimumab, CP-675,206).). In an embodiment, the agent is anantibody or antibody fragment that binds to TIM3. In an embodiment, theagent is an antibody or antibody fragment that binds to LAG3.

PD-1 is an inhibitory member of the CD28 family of receptors that alsoincludes CD28, CTLA-4, ICOS, and BTLA. PD-1 is expressed on activated Bcells, T cells and myeloid cells (Agata et al. 1996 Int. Immunol8:765-75). Two ligands for PD-1, PD-L1 and PD-L2 have been shown todownregulate T cell activation upon binding to PD-1 (Freeman et a. 2000J Exp Med 192:1027-34; Latchman et al. 2001 Nat Immunol 2:261-8; Carteret al. 2002 Eur J Immunol 32:634-43). PD-L1 is abundant in human cancers(Dong et al. 2003 J Mol Med 81:281-7; Blank et al. 2005 Cancer Immunol.Immunother 54:307-314; Konishi et al. 2004 Clin Cancer Res 10:5094).Immune suppression can be reversed by inhibiting the local interactionof PD-1 with PD-L1. Antibodies, antibody fragments, and other inhibitorsof PD-1, PD-L1 and PD-L2 are available in the art and may be usedcombination with a CAR of the present invention described herein. Forexample, nivolumab (also referred to as BMS-936558 or MDX1106;Bristol-Myers Squibb) is a fully human IgG4 monoclonal antibody whichspecifically blocks PD-1. Nivolumab (clone 5C4) and other humanmonoclonal antibodies that specifically bind to PD-1 are disclosed inU.S. Pat. No. 8,008,449 and WO2006/121168. Pidilizumab (CT-011; CureTech) is a humanized IgGlk monoclonal antibody that binds to PD-1.Pidilizumab and other humanized anti-PD-1 monoclonal antibodies aredisclosed in WO2009/101611. Pembrolizumab (formerly known aslambrolizumab, and also referred to as MK03475; Merck) is a humanizedIgG4 monoclonal antibody that binds to PD-1. Pembrolizumab and otherhumanized anti-PD-1 antibodies are disclosed in U.S. Pat. No. 8,354,509and WO2009/114335. MEDI4736 (Medimmune) is a human monoclonal antibodythat binds to PDL1, and inhibits interaction of the ligand with PD1.MDPL3280A (Genentech/Roche) is a human Fc optimized IgG1 monoclonalantibody that binds to PD-L1. MDPL3280A and other human monoclonalantibodies to PD-L1 are disclosed in U.S. Pat. No. 7,943,743 and U.SPublication No.: 20120039906. Other anti-PD-L1 binding agents includeYW243.55.570 (heavy and light chain variable regions are shown in SEQ IDNOs 20 and 21 in WO2010/077634) and MDX-1 105 (also referred to asBMS-936559, and, e.g., anti-PD-L1 binding agents disclosed inWO2007/005874). AMP-224 (B7-DCIg; Amplimmune; e.g., disclosed inWO2010/027827 and WO2011/066342), is a PD-L2 Fc fusion soluble receptorthat blocks the interaction between PD-1 and B7-H1. Other anti-PD-1antibodies include AMP 514 (Amplimmune), among others, e.g., anti-PD-1antibodies disclosed in U.S. Pat. No. 8,609,089, US 2010028330, and/orUS 20120114649.

TIM3 (T cell immunoglobulin-3) also negatively regulates T cellfunction, particularly in IFN-g-secreting CD4+T helper 1 and CD8+Tcytotoxic 1 cells, and plays a critical role in T cell exhaustion.Inhibition of the interaction between TIM3 and its ligands, e.g.,galectin-9 (Ga19), phosphotidylserine (PS), and HMGB1, can increaseimmune response. Antibodies, antibody fragments, and other inhibitors ofTIM3 and its ligands are available in the art and may be usedcombination with a CD19 CAR described herein. For example, antibodies,antibody fragments, small molecules, or peptide inhibitors that targetTIM3 binds to the IgV domain of TIM3 to inhibit interaction with itsligands. Antibodies and peptides that inhibit TIM3 are disclosed inWO2013/006490 and US20100247521. Other anti-TIM3 antibodies includehumanized versions of RMT3-23 (disclosed in Ngiow et al., 2011, CancerRes, 71:3540-3551), and clone 8B.2C12 (disclosed in Monney et al., 2002,Nature, 415:536-541). Bi-specific antibodies that inhibit TIM3 and PD-1are disclosed in US20130156774.

In other embodiments, the agent which enhances the activity of aCAR-expressing cell is a CEACAM inhibitor (e.g., CEACAM-1, CEACAM-3,and/or CEACAM-5 inhibitor). In one embodiment, the inhibitor of CEACAMis an anti-CEACAM antibody molecule. Exemplary anti-CEACAM-1 antibodiesare described in WO 2010/125571, WO 2013/082366 WO 2014/059251 and WO2014/022332, e.g., a monoclonal antibody 34B1, 26H7, and 5F4; or arecombinant form thereof, as described in, e.g., US 2004/0047858, U.S.Pat. No. 7,132,255 and WO 99/052552. In other embodiments, theanti-CEACAM antibody binds to CEACAM-5 as described in, e.g., Zheng etal. PLoS One. 2010 Sep. 2; 5(9). pii: e12529(DOI:10:1371/journal.pone.0021146), or crossreacts with CEACAM-1 andCEACAM-5 as described in, e.g., WO 2013/054331 and US 2014/0271618.

Without wishing to be bound by theory, carcinoembryonic antigen celladhesion molecules (CEACAM), such as CEACAM-1 and CEACAM-5, are believedto mediate, at least in part, inhibition of an anti-tumor immuneresponse (see e.g., Markel et al. J Immunol. 2002 Mar. 15;168(6):2803-10; Markel et al. J Immunol. 2006 Nov. 1; 177(9):6062-71;Markel et al. Immunology. 2009 February; 126(2):186-200; Markel et al.Cancer Immunol Immunother. 2010 February; 59(2):215-30; Ortenberg et al.Mol Cancer Ther. 2012 June; 11(6):1300-10; Stern et al. J Immunol. 2005Jun. 1; 174(11):6692-701; Zheng et al. PLoS One. 2010 Sep. 2; 5(9). pii:e12529). For example, CEACAM-1 has been described as a heterophilicligand for TIM-3 and as playing a role in TIM-3-mediated T celltolerance and exhaustion (see e.g., WO 2014/022332; Huang, et al. (2014)Nature doi:10.1038/nature13848). In embodiments, co-blockade of CEACAM-1and TIM-3 has been shown to enhance an anti-tumor immune response inxenograft colorectal cancer models (see e.g., WO 2014/022332; Huang, etal. (2014), supra). In other embodiments, co-blockade of CEACAM-1 andPD-1 reduce T cell tolerance as described, e.g., in WO 2014/059251.Thus, CEACAM inhibitors can be used with the other immunomodulatorsdescribed herein (e.g., anti-PD-1 and/or anti-TIM-3 inhibitors) toenhance an immune response against a cancer, e.g., a melanoma, a lungcancer (e.g., NSCLC), a bladder cancer, a colon cancer an ovariancancer, and other cancers as described herein

LAG3 (lymphocyte activation gene-3 or CD223) is a cell surface moleculeexpressed on activated T cells and B cells that has been shown to play arole in CD8+ T cell exhaustion. Antibodies, antibody fragments, andother inhibitors of LAG3 and its ligands are available in the art andmay be used combination with a CD19 CAR described herein. For example,BMS-986016 (Bristol-Myers Squib) is a monoclonal antibody that targetsLAG3. IMP701 (Immutep) is an antagonist LAG3 antibody and IMP731(Immutep and GlaxoSmithKline) is a depleting LAG3 antibody. Other LAG3inhibitors include IMP321 (Immutep), which is a recombinant fusionprotein of a soluble portion of LAG3 and Ig that binds to WIC class IImolecules and activates antigen presenting cells (APC). Other antibodiesare disclosed, e.g., in WO2010/019570.

In some embodiments, the agent which enhances the activity of aCAR-expressing cell can be, e.g., a fusion protein comprising a firstdomain and a second domain, wherein the first domain is an inhibitorymolecule, or fragment thereof, and the second domain is a polypeptidethat is associated with a positive signal, e.g., a polypeptide comrpsingan antracellular signaling domain as described herein. In someembodiments, the polypeptide that is associated with a positive signalcan include a costimulatory domain of CD28, CD27, ICOS, e.g., anintracellular signaling domain of CD28, CD27 and/or ICOS, and/or aprimary signaling domain, e.g., of CD3 zeta, e.g., described herein. Inone embodiment, the fusion protein is expressed by the same cell thatexpressed the CAR. In another embodiment, the fusion protein isexpressed by a cell, e.g., a T cell that does not express a mesothelinCAR.

In one embodiment, the agent which enhances activity of a CAR-expressingcell described herein is miR-17-92.

Combination with a Low Dose of an mTOR Inhibitor

In one embodiment, the cells expressing a CAR molecule, e.g., a CARmolecule described herein, are administered in combination with a low,immune enhancing dose of an mTOR inhibitor.

In an embodiment, a dose of an mTOR inhibitor is associated with, orprovides, mTOR inhibition of at least 5 but no more than 90%, at least10 but no more than 90%, at least 15, but no more than 90%, at least 20but no more than 90%, at least 30 but no more than 90%, at least 40 butno more than 90%, at least 50 but no more than 90%, at least 60 but nomore than 90%, or at least 70 but no more than 90%.

In an embodiment, a dose of an mTOR inhibitor is associated with, orprovides, mTOR inhibition of at least 5 but no more than 80%, at least10 but no more than 80%, at least 15, but no more than 80%, at least 20but no more than 80%, at least 30 but no more than 80%, at least 40 butno more than 80%, at least 50 but no more than 80%, or at least 60 butno more than 80%.

In an embodiment, a dose of an mTOR inhibitor is associated with, orprovides, mTOR inhibition of at least 5 but no more than 70%, at least10 but no more than 70%, at least 15, but no more than 70%, at least 20but no more than 70%, at least 30 but no more than 70%, at least 40 butno more than 70%, or at least 50 but no more than 70%.

In an embodiment, a dose of an mTOR inhibitor is associated with, orprovides, mTOR inhibition of at least 5 but no more than 60%, at least10 but no more than 60%, at least 15, but no more than 60%, at least 20but no more than 60%, at least 30 but no more than 60%, or at least 40but no more than 60%.

In an embodiment, a dose of an mTOR inhibitor is associated with, orprovides, mTOR inhibition of at least 5 but no more than 50%, at least10 but no more than 50%, at least 15, but no more than 50%, at least 20but no more than 50%, at least 30 but no more than 50%, or at least 40but no more than 50%.

In an embodiment, a dose of an mTOR inhibitor is associated with, orprovides, mTOR inhibition of at least 5 but no more than 40%, at least10 but no more than 40%, at least 15, but no more than 40%, at least 20but no more than 40%, at least 30 but no more than 40%, or at least 35but no more than 40%.

In an embodiment, a dose of an mTOR inhibitor is associated with, orprovides, mTOR inhibition of at least 5 but no more than 30%, at least10 but no more than 30%, at least 15, but no more than 30%, at least 20but no more than 30%, or at least 25 but no more than 30%.

In an embodiment, a dose of an mTOR inhibitor is associated with, orprovides, mTOR inhibition of at least 1, 2, 3, 4 or 5 but no more than20%, at least 1, 2, 3, 4 or 5 but no more than 30%, at least 1, 2, 3, 4or 5, but no more than 35, at least 1, 2, 3, 4 or 5 but no more than40%, or at least 1, 2, 3, 4 or 5 but no more than 45%.

In an embodiment, a dose of an mTOR inhibitor is associated with, orprovides, mTOR inhibition of at least 1, 2, 3, 4 or 5 but no more than90%.

As is discussed herein, the extent of mTOR inhibition can be expressedas the extent of P70 S6 inhibition, e.g., the extent of mTOR inhibitioncan be determined by the level of decrease in P70 S6 activity, e.g., bythe decrease in phosphorylation of a P70 S6 substrate. The level of mTORinhibition can be evaluated by a method described herein, e.g. by theBoulay assay.

Exemplary Mtor Inhibitors

As used herein, the term “mTOR inhibitor” refers to a compound orligand, or a pharmaceutically acceptable salt thereof, which inhibitsthe mTOR kinase in a cell. In an embodiment an mTOR inhibitor is anallosteric inhibitor. In an embodiment an mTOR inhibitor is a catalyticinhibitor.

Allosteric mTOR inhibitors include the neutral tricyclic compoundrapamycin (sirolimus), rapamycin-related compounds, that is compoundshaving structural and functional similarity to rapamycin including,e.g., rapamycin derivatives, rapamycin analogs (also referred to asrapalogs) and other macrolide compounds that inhibit mTOR activity.

Rapamycin is a known macrolide antibiotic produced by Streptomyceshygroscopicus having the structure shown in Formula A.

See, e.g., McAlpine, J. B., et al., J. Antibiotics (1991) 44: 688;Schreiber, S. L., et al., J. Am. Chem. Soc. (1991) 113: 7433; U.S. Pat.No. 3,929,992. There are various numbering schemes proposed forrapamycin. To avoid confusion, when specific rapamycin analogs are namedherein, the names are given with reference to rapamycin using thenumbering scheme of formula A.

Rapamycin analogs useful in the invention are, for example,0-substituted analogs in which the hydroxyl group on the cyclohexyl ringof rapamycin is replaced by OR₁ in which R₁ is hydroxyalkyl,hydroxyalkoxyalkyl, acylaminoalkyl, or aminoalkyl; e.g. RAD001, alsoknown as, everolimus as described in U.S. Pat. No. 5,665,772 andWO94/09010 the contents of which are incorporated by reference. Othersuitable rapamycin analogs include those substituted at the 26- or28-position. The rapamycin analog may be an epimer of an analogmentioned above, particularly an epimer of an analog substituted inposition 40, 28 or 26, and may optionally be further hydrogenated, e.g.as described in U.S. Pat. No. 6,015,815, WO95/14023 and WO99/15530 thecontents of which are incorporated by reference, e.g. ABT578 also knownas zotarolimus or a rapamycin analog described in U.S. Pat. No.7,091,213, WO98/02441 and WO01/14387 the contents of which areincorporated by reference, e.g. AP23573 also known as ridaforolimus.

Examples of rapamycin analogs suitable for use in the present inventionfrom U.S. Pat. No. 5,665,772 include, but are not limited to,40-O-benzyl-rapamycin, 40-O-(4′-hydroxymethyl)benzyl-rapamycin,40-O-[4′-(1,2-dihydroxyethyl)]benzyl-rapamycin, 40-O-allyl-rapamycin,40-O-[3′-(2,2-dimethyl-1,3-dioxolan-4(S)-yl)-prop-2′-en-1′-yl]-rapamycin,(2′E,4'S)-40-O-(4′,5′-dihydroxypent-2′-en-1′-yl)-rapamycin,40-O-(2-hydroxy)ethoxycarbonylmethyl-rapamycin,40-O-(2-hydroxy)ethyl-rapamycin, 40-O-(3-hydroxy)propyl-rapamycin,40-O-(6-hydroxy)hexyl-rapamycin,40-O-[2-(2-hydroxy)ethoxy]ethyl-rapamycin,40-O-[(3S)-2,2-dimethyldioxolan-3-yl]methyl-rapamycin,40-O-[(2S)-2,3-dihydroxyprop-1-yl]-rapamycin,40-O-(2-acetoxy)ethyl-rapamycin, 40-O-(2-nicotinoyloxy)ethyl-rapamycin,40-O-[2-(N-morpholino)acetoxy]ethyl-rapamycin,40-O-(2-N-imidazolylacetoxy)ethyl-rapamycin,40-O-[2-(N-methyl-N′-piperazinyl)acetoxy]ethyl-rapamycin,39-O-desmethyl-39,40-O,O-ethylene-rapamycin,(26R)-26-dihydro-40-O-(2-hydroxy)ethyl-rapamycin,40-O-(2-aminoethyl)-rapamycin, 40-O-(2-acetaminoethyl)-rapamycin,40-O-(2-nicotinamidoethyl)-rapamycin,40-O-(2-(N-methyl-imidazo-2′-ylcarbethoxamido)ethyl)-rapamycin,40-O-(2-ethoxycarbonylaminoethyl)-rapamycin,40-O-(2-tolylsulfonamidoethyl)-rapamycin and40-O-[2-(4′,5′-dicarboethoxy-1′,2′,3′-triazol-1′-yl)-ethyl]-rapamycin.

Other rapamycin analogs useful in the present invention are analogswhere the hydroxyl group on the cyclohexyl ring of rapamycin and/or thehydroxy group at the 28 position is replaced with an hydroxyester groupare known, for example, rapamycin analogs found in U.S. RE44,768, e.g.temsirolimus.

Other rapamycin analogs useful in the preset invention include thosewherein the methoxy group at the 16 position is replaced with anothersubstituent, preferably (optionally hydroxy-substituted) alkynyloxy,benzyl, orthomethoxybenzyl or chlorobenzyl and/or wherein the mexthoxygroup at the 39 position is deleted together with the 39 carbon so thatthe cyclohexyl ring of rapamycin becomes a cyclopentyl ring lacking the39 position methyoxy group; e.g. as described in WO95/16691 andWO96/41807 the contents of which are incorporated by reference. Theanalogs can be further modified such that the hydroxy at the 40-positionof rapamycin is alkylated and/or the 32-carbonyl is reduced.

Rapamycin analogs from WO95/16691 include, but are not limited to,16-demthoxy-16-(pent-2-ynyl)oxy-rapamycin,16-demthoxy-16-(but-2-ynyl)oxy-rapamycin,16-demthoxy-16-(propargyl)oxy-rapamycin,16-demethoxy-16-(4-hydroxy-but-2-ynyl)oxy-rapamycin,16-demthoxy-16-benzyloxy-40-O-(2-hydroxyethyl)-rapamycin,16-demthoxy-16-benzyloxy-rapamycin,16-demethoxy-16-ortho-methoxybenzyl-rapamycin,16-demethoxy-40-O-(2-methoxyethyl)-16-pent-2-ynyl)oxy-rapamycin,39-demethoxy-40-desoxy-39-formyl-42-nor-rapamycin,39-demethoxy-40-desoxy-39-hydroxymethyl-42-nor-rapamycin,39-demethoxy-40-desoxy-39-carboxy-42-nor-rapamycin,39-demethoxy-40-desoxy-39-(4-methyl-piperazin-1-yl)carbonyl-42-nor-rapamycin,39-demethoxy-40-desoxy-39-(morpholin-4-yl)carbonyl-42-nor-rapamycin,39-demethoxy-40-desoxy-39-[N-methyl,N-(2-pyridin-2-yl-ethyl)]carbamoyl-42-nor-rapamycin and39-demethoxy-40-desoxy-39-(p-toluenesulfonylhydrazonomethyl)-42-nor-rapamycin.

Rapamycin analogs from WO96/41807 include, but are not limited to,32-deoxo-rapamycin, 16-O-pent-2-ynyl-32-deoxo-rapamycin,16-O-pent-2-ynyl-32-deoxo-40-O-(2-hydroxy-ethyl)-rapamycin,16-O-pent-2-ynyl-32-(S)-dihydro-40-O-(2-hydroxyethyl)-rapamycin,32(S)-dihydro-40-O-(2-methoxy)ethyl-rapamycin and32(S)-dihydro-40-O-(2-hydroxyethyl)-rapamycin.

Another suitable rapamycin analog is umirolimus as described inUS2005/0101624 the contents of which are incorporated by reference.

RAD001, otherwise known as everolimus (Afinitor®), has the chemical name(1R,9S,12S,15R,16E,18R,19R,21R,23S,24E,26E,28E,30S,32S,35R)-1,18-dihydroxy-12-{(1R)-2-[(1S,3R,4R)-4-(2-hydroxyethoxy)-3-methoxycyclohexyl]-1-methylethyl}-19,30-dimethoxy-15,17,21,23,29,35-hexamethyl-11,36-dioxa-4-aza-tricyclo[30.3.1.04,9]hexatriaconta-16,24,26,28-tetraene-2,3,10,14,20-pentaone

Further examples of allosteric mTOR inhibitors include sirolimus(rapamycin, AY-22989),40-[3-hydroxy-2-(hydroxymethyl)-2-methylpropanoate]-rapamycin (alsocalled temsirolimus or CCI-779) and ridaforolimus (AP-23573/MK-8669).Other examples of allosteric mTor inhibtors include zotarolimus (ABT578)and umirolimus.

Alternatively or additionally, catalytic, ATP-competitive mTORinhibitors have been found to target the mTOR kinase domain directly andtarget both mTORC1 and mTORC2. These are also more effective inhibitorsof mTORC1 than such allosteric mTOR inhibitors as rapamycin, becausethey modulate rapamycin-resistant mTORC1 outputs such as 4EBP1-T37/46phosphorylation and cap-dependent translation.

Catalytic inhibitors include: BEZ235 or2-methyl-2-[4-(3-methyl-2-oxo-8-quinolin-3-yl-2,3-dihydro-imidazo[4,5-c]quinolin-1-yl)-phenyl]-propionitrile,or the monotosylate salt form. the synthesis of BEZ235 is described inWO2006/122806; CCG168 (otherwise known as AZD-8055, Chresta, C. M., etal., Cancer Res, 2010, 70(1), 288-298) which has the chemical name{5-[2,4-bis-((S)-3-methyl-morpholin-4-yl)-pyrido[2,3d]pyrimidin-7-yl]-2-methoxy-phenyl}-methanol;3-[2,4-bis[(3S)-3-methylmorpholin-4-yl]pyrido[2,3-d]pyrimidin-7-yl]-N-methylbenzamide(WO09104019);3-(2-aminobenzo[d]oxazol-5-yl)-1-isopropyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine(WO10051043 and WO2013023184); AN-(3-(N-(3-((3,5-dimethoxyphenyl)amino)quinoxaline-2-yl)sulfamoyl)phenyl)-3-methoxy-4-methylbenzamide(WO07044729 and WO12006552); PKI-587 (Venkatesan, A. M., J. Med. Chem.,2010, 53, 2636-2645) which has the chemical name1-[4-[4-(dimethylamino)piperidine-1-carbonyl]phenyl]-3-[4-(4,6-dimorpholino-1,3,5-triazin-2-yl)phenyl]urea;GSK-2126458 (ACS Med. Chem. Lett., 2010, 1, 39-43) which has thechemical name2,4-difluoro-N-{2-methoxy-5-[4-(4-pyridazinyl)-6-quinolinyl]-3-pyridinyl}benzenesulfonamide;5-(9-isopropyl-8-methyl-2-morpholino-9H-purin-6-yl)pyrimidin-2-amine(WO10114484);(E)-N-(8-(6-amino-5-(trifluoromethyl)pyridin-3-yl)-1-(6-(2-cyanopropan-2-yl)pyridin-3-yl)-3-methyl-1H-imidazo[4,5-c]quinolin-2(3H)-ylidene)cyanamide(WO12007926).

Further examples of catalytic mTOR inhibitors include8-(6-methoxy-pyridin-3-yl)-3-methyl-1-(4-piperazin-1-yl-3-trifluoromethyl-phenyl)-1,3-dihydro-imidazo[4,5-c]quinolin-2-one(WO2006/122806) and Ku-0063794 (Garcia-Martinez J M, et al., Biochem J.,2009, 421(1), 29-42. Ku-0063794 is a specific inhibitor of the mammaliantarget of rapamycin (mTOR).) WYE-354 is another example of a catalyticmTor inhibitor (Yu K, et al. (2009). Biochemical, Cellular, and In vivoActivity of Novel ATP-Competitive and Selective Inhibitors of theMammalian Target of Rapamycin. Cancer Res. 69(15): 6232-6240).

mTOR inhibitors useful according to the present invention also includeprodrugs, derivatives, pharmaceutically acceptable salts, or analogsthereof of any of the foregoing.

mTOR inhibitors, such as RAD001, may be formulated for delivery based onwell-established methods in the art based on the particular dosagesdescribed herein. In particular, U.S. Pat. No. 6,004,973 (incorporatedherein by reference) provides examples of formulations useable with themTOR inhibitors described herein.

Evaluation of Mtor Inhibition

mTOR phosphorylates the kinase P70 S6, thereby activating P70 S6 kinaseand allowing it to phosphorylate its substrate. The extent of mTORinhibition can be expressed as the extent of P70 S6 kinase inhibition,e.g., the extent of mTOR inhibition can be determined by the level ofdecrease in P70 S6 kinase activity, e.g., by the decrease inphosphorylation of a P70 S6 kinase substrate. One can determine thelevel of mTOR inhibition, by measuring P70 S6 kinase activity (theability of P70 S6 kinase to phsophorylate a substrate), in the absenceof inhibitor, e.g., prior to administration of inhibitor, and in thepresences of inhibitor, or after the administration of inhibitor. Thelevel of inhibition of P70 S6 kinase gives the level of mTOR inhibition.Thus, if P70 S6 kinase is inhibited by 40%, mTOR activity, as measuredby P70 S6 kinase activity, is inhibited by 40%. The extent or level ofinhibition referred to herein is the average level of inhibition overthe dosage interval. By way of example, if the inhibitor is given onceper week, the level of inhibition is given by the average level ofinhibition over that interval, namely a week.

Boulay et al., Cancer Res, 2004, 64:252-61, hereby incorporated byreference, teaches an assay that can be used to assess the level of mTORinhibition (referred to herein as the Boulay assay). In an embodiment,the assay relies on the measurement of P70 S6 kinase activity frombiological samples before and after administration of an mTOR inhibitor,e.g., RAD001. Samples can be taken at preselected times after treatmentwith an mTOR ihibitor, e.g., 24, 48, and 72 hours after treatment.Biological samples, e.g., from skin or peripheral blood mononuclearcells (PBMCs) can be used. Total protein extracts are prepared from thesamples. P70 S6 kinase is isolated from the protein extracts byimmunoprecipitation using an antibody that specifically recognizes theP70 S6 kinase. Activity of the isolated P70 S6 kinase can be measured inan in vitro kinase assay. The isolated kinase can be incubated with 40Sribosomal subunit substrates (which is an endogenous substrate of P70 S6kinase) and gamma-³²P under conditions that allow phosphorylation of thesubstrate. Then the reaction mixture can be resolved on an SDS-PAGE gel,and ³²P signal analyzed using a Phosphorlmager. A ³²P signalcorresponding to the size of the 40S ribosomal subunit indicatesphosphorylated substrate and the activity of P70 S6 kinase. Increasesand decreases in kinase activity can be calculated by quantifying thearea and intensity of the ³²P signal of the phosphorylated substrate(e.g., using ImageQuant, Molecular Dynamics), assigning arbitrary unitvalues to the quantified signal, and comparing the values from afteradministration with values from before administration or with areference value. For example, percent inhibition of kinase activity canbe calculated with the following formula: 1-(value obtained afteradministration/value obtained before administration)×100. As describedabove, the extent or level of inhibition referred to herein is theaverage level of inhibition over the dosage interval.

Methods for the evaluation of kinase activity, e.g., P70 S6 kinaseactivity, are also provided in U.S. Pat. No. 7,727,950, herebyincorporated by reference.

The level of mTOR inhibition can also be evaluated by a change in theration of PD1 negative to PD1 positive T cells. T cells from peripheralblood can be identified as PD1 negative or positive by art-knownmethods.

Low-Dose mTOR Inhibitors

Methods described herein use low, immune enhancing, dose mTORinhibitors, doses of mTOR inhibitors, e.g., allosteric mTOR inhibitors,including rapalogs such as RAD001. In contrast, levels of inhibitor thatfully or near fully inhibit the mTOR pathway are immunosuppressive andare used, e.g., to prevent organ transplant rejection. In addition, highdoses of rapalogs that fully inhibit mTOR also inhibit tumor cell growthand are used to treat a variety of cancers (See, e.g., Antineoplasticeffects of mammalian target of rapamycine inhibitors. Salvadori M. WorldJ Transplant. 2012 Oct. 24; 2(5):74-83; Current and Future TreatmentStrategies for Patients with Advanced Hepatocellular Carcinoma: Role ofmTOR Inhibition. Finn R S. Liver Cancer. 2012 November; 1(3-4):247-256;Emerging Signaling Pathways in Hepatocellular Carcinoma. Moeini A,Cornelia H, Villanueva A. Liver Cancer. 2012 September; 1(2):83-93;Targeted cancer therapy—Are the days of systemic chemotherapy numbered?Joo W D, Visintin I, Mor G. Maturitas. 2013 Sep. 20; Role of natural andadaptive immunity in renal cell carcinoma response to VEGFR-TKIs andmTOR inhibitor. Santoni M, Berardi R, Amantini C, Burattini L, SantiniD, Santoni G, Cascinu S. Int J Cancer. 2013 Oct. 2).

The present invention is based, at least in part, on the surprisingfinding that doses of mTOR inhibitors well below those used in currentclinical settings had a superior effect in increasing an immune responsein a subject and increasing the ratio of PD-1 negative T cells/PD-1positive T cells. It was surprising that low doses of mTOR inhibitors,producing only partial inhibition of mTOR activity, were able toeffectively improve immune responses in human subjects and increase theratio of PD-1 negative T cells/PD-1 positive T cells.

Alternatively, or in addition, without wishing to be bound by anytheory, it is believed that low, a low, immune enhancing, dose of anmTOR inhibitor can increase naive T cell numbers, e.g., at leasttransiently, e.g., as compared to a non-treated subject. Alternativelyor additionally, again while not wishing to be bound by theory, it isbelieved that treatment with an mTOR inhibitor after a sufficient amountof time or sufficient dosing results in one or more of the following:

an increase in the expression of one or more of the following markers:CD62L^(high), CD127^(high), CD27⁺, and BCL2, e.g., on memory T cells,e.g., memory T cell precursors;

a decrease in the expression of KLRG1, e.g., on memory T cells, e.g.,memory T cell precursors; and

an increase in the number of memory T cell precursors, e.g., cells withany one or combination of the following characteristics: increasedCD62L^(high), increased CD127^(high), increased CD27⁺, decreased KLRG1,and increased BCL2;

and wherein any of the changes described above occurs, e.g., at leasttransiently, e.g., as compared to a non-treated subject (Araki, K et al.(2009) Nature 460:108-112). Memory T cell precursors are memory T cellsthat are early in the differentiation program. For example, memory Tcells have one or more of the following characteristics: increasedCD62L^(high), increased CD127^(high), increased CD27⁺, decreased KLRG1,and/or increased BCL2.

In an embodiment, the invention relates to a composition, or dosageform, of an mTOR inhibitor, e.g., an allosteric mTOR inhibitor, e.g., arapalog, rapamycin, or RAD001, or a catalytic mTOR inhibitor, which,when administered on a selected dosing regimen, e.g., once daily or onceweekly, is associated with: a level of mTOR inhibition that is notassociated with complete, or significant immune suppression, but isassociated with enhancement of the immune response.

An mTOR inhibitor, e.g., an allosteric mTOR inhibitor, e.g., a rapalog,rapamycin, or RAD001, or a catalytic mTOR inhibitor, can be provided ina sustained relase formulation. Any of the compositions or unit dosageforms described herein can be provided in a sustained releaseformulation. In some embodiments, a sustained release formulation willhave lower bioavailability than an immediate release formulation. E.g.,in embodiments, to attain a similar therapeutic effect of an immediaterelease forlation a sustained release formulation will have from about 2to about 5, about 2.5 to about 3.5, or about 3 times the amount ofinhibitor provided in the immediate release formulation.

In an embodiment, immediate release forms, e.g., of RAD001, typicallyused for one administration per week, having 0.1 to 20, 0.5 to 10, 2.5to 7.5, 3 to 6, or about 5, mgs per unit dosage form, are provided. Foronce per week administrations, these immediate release formulationscorrespond to sustained release forms, having, respectively, 0.3 to 60,1.5 to 30, 7.5 to 22.5, 9 to 18, or about 15 mgs of an mTOR inhibitor,e.g., an allosteric mTOR inhibitor, e.g., rapamycin or RAD001. Inembodiments both forms are administered on a once/week basis.

In an embodiment, immediate release forms, e.g., of RAD001, typicallyused for one administration per day, having having 0.005 to 1.5, 0.01 to1.5, 0.1 to 1.5, 0.2 to 1.5, 0.3 to 1.5, 0.4 to 1.5, 0.5 to 1.5, 0.6 to1.5, 0.7 to 1.5, 0.8 to 1.5, 1.0 to 1.5, 0.3 to 0.6, or about 0.5 mgsper unit dosage form, are provided. For once per day administrations,these immediate release forms correspond to sustained release forms,having, respectively, 0.015 to 4.5, 0.03 to 4.5, 0.3 to 4.5, 0.6 to 4.5,0.9 to 4.5, 1.2 to 4.5, 1.5 to 4.5, 1.8 to 4.5, 2.1 to 4.5, 2.4 to 4.5,3.0 to 4.5, 0.9 to 1.8, or about 1.5 mgs of an mTOR inhibitor, e.g., anallosteric mTOR inhibitor, e.g., rapamycin or RAD001. For once per weekadministrations, these immediate release forms correspond to sustainedrelease forms, having, respectively, 0.1 to 30, 0.2 to 30, 2 to 30, 4 to30, 6 to 30, 8 to 30, 10 to 30, 1.2 to 30, 14 to 30, 16 to 30, 20 to 30,6 to 12, or about 10 mgs of an mTOR inhibitor, e.g., an allosteric mTORinhibitor, e.g., rapamycin or RAD001.

In an embodiment, immediate release forms, e.g., of RAD001, typicallyused for one administration per day, having having 0.01 to 1.0 mgs perunit dosage form, are provided. For once per day administrations, theseimmediate release forms correspond to sustained release forms, having,respectively, 0.03 to 3 mgs of an mTOR inhibitor, e.g., an allostericmTOR inhibitor, e.g., rapamycin or RAD001. For once per weekadministrations, these immediate release forms correspond to sustainedrelease forms, having, respectively, 0.2 to 20 mgs of an mTOR inhibitor,e.g., an allosteric mTOR inhibitor, e.g., rapamycin or RAD001.

In an embodiment, immediate release forms, e.g., of RAD001, typicallyused for one administration per week, having having 0.5 to 5.0 mgs perunit dosage form, are provided. For once per week administrations, theseimmediate release forms correspond to sustained release forms, having,respectively, 1.5 to 15 mgs of an mTOR inhibitor, e.g., an allostericmTOR inhibitor, e.g., rapamycin or RAD001.

As described above, one target of the mTOR pathway is the P70 S6 kinase.Thus, doses of mTOR inhibitors which are useful in the methods andcompositions described herein are those which are sufficient to achieveno greater than 80% inhibition of P70 S6 kinase activity relative to theactivity of the P70 S6 kinase in the absence of an mTOR inhibitor, e.g.,as measured by an assay described herein, e.g., the Boulay assay. In afurther aspect, the invention provides an amount of an mTOR inhibitorsufficient to achieve no greater than 38% inhibition of P70 S6 kinaseactivity relative to P70 S6 kinase activity in the absence of an mTORinhibitor.

In one aspect the dose of mTOR inhibitor useful in the methods andcompositions of the invention is sufficient to achieve, e.g., whenadministered to a human subject, 90+/−5% (i.e., 85-95%), 89+/−5%,88+/−5%, 87+/−5%, 86+/−5%, 85+/−5%, 84+/−5%, 83+/−5%, 82+/−5%, 81+/−5%,80+/−5%, 79+/−5%, 78+/−5%, 77+/−5%, 76+/−5%, 75+/−5%, 74+/−5%, 73+/−5%,72+/−5%, 71+/−5%, 70+/−5%, 69+/−5%, 68+/−5%, 67+/−5%, 66+/−5%, 65+/−5%,64+/−5%, 63+/−5%, 62+/−5%, 61+/−5%, 60+/−5%, 59+/−5%, 58+/−5%, 57+/−5%,56+/−5%, 55+/−5%, 54+/−5%, 54+/−5%, 53+/−5%, 52+/−5%, 51+/−5%, 50+/−5%,49+/−5%, 48+/−5%, 47+/−5%, 46+/−5%, 45+/−5%, 44+/−5%, 43+/−5%, 42+/−5%,41+/−5%, 40+/−5%, 39+/−5%, 38+/−5%, 37+/−5%, 36+/−5%, 35+/−5%, 34+/−5%,33+/−5%, 32+/−5%, 31+/−5%, 30+/−5%, 29+/−5%, 28+/−5%, 27+/−5%, 26+/−5%,25+/−5%, 24+/−5%, 23+/−5%, 22+/−5%, 21+/−5%, 20+/−5%, 19+/−5%, 18+/−5%,17+/−5%, 16+/−5%, 15+/−5%, 14+/−5%, 13+/−5%, 12+/−5%, 11+/−5%, or10+/−5%, inhibition of P70 S6 kinase activity, e.g., as measured by anassay described herein, e.g., the Boulay assay.

P70 S6 kinase activity in a subject may be measured using methods knownin the art, such as, for example, according to the methods described inU.S. Pat. No. 7,727,950, by immunoblot analysis of phosphoP70 S6K levelsand/or phosphoP70 S6 levels or by in vitro kinase activity assays.

As used herein, the term “about” in reference to a dose of mTORinhibitor refers to up to a +/−10% variability in the amount of mTORinhibitor, but can include no variability around the stated dose.

In some embodiments, the invention provides methods comprisingadministering to a subject an mTOR inhibitor, e.g., an allostericinhibitor, e.g., RAD001, at a dosage within a target trough level. Insome embodiments, the trough level is significantly lower than troughlevels associated with dosing regimens used in organ transplant andcancer patients. In an embodiment mTOR inhibitor, e.g., RAD001, orrapamycin, is administerd to result in a trough level that is less than½, ¼, 1/10, or 1/20 of the trough level that results inimmunosuppression or an anticancer effect. In an embodiment mTORinhibitor, e.g., RAD001, or rapamycin, is administerd to result in atrough level that is less than ½, ¼, 1/10, or 1/20 of the trough levelprovided on the FDA approved packaging insert for use inimmunosuppression or an anticancer indications.

In an embodiment a method disclosed herein comprises administering to asubject an mTOR inhibitor, e.g., an allosteric inhibitor, e.g., RAD001,at a dosage that provides a target trough level of 0.1 to 10 ng/ml, 0.1tO 5 ng/ml, 0.1 to 3 ng/ml, 0.1 to 2 ng/ml, or 0.1 to 1 ng/ml.

In an embodiment a method disclosed herein comprises administering to asubject an mTOR inhibitor, e.g., an allosteric inhibitor, e.g., RAD001,at a dosage that provides a target trough level of 0.2 to 10 ng/ml, 0.2to 5 ng/ml, 0.2 to 3 ng/ml, 0.2 to 2 ng/ml, or 0.2 to 1 ng/ml.

In an embodiment a method disclosed herein comprises administering to asubject an mTOR inhibitor, e.g. an, allosteric inhibitor, e.g., RAD001,at a dosage that provides a target trough level of 0.3 to 10 ng/ml, 0.3to 5 ng/ml, 0.3 to 3 ng/ml, 0.3 to 2 ng/ml, or 0.3 to 1 ng/ml.

In an embodiment a method disclosed herein comprises administering to asubject an mTOR inhibitor, e.g., an allosteric inhibitor, e.g., RAD001,at a dosage that provides a target trough level of 0.4 to 10 ng/ml, 0.4to 5 ng/ml, 0.4 to 3 ng/ml, 0.4 to 2 ng/ml, or 0.4 to 1 ng/ml.

In an embodiment a method disclosed herein comprises administering to asubject an mTOR inhibitor, e.g., an allosteric inhibitor, e.g., RAD001,at a dosage that provides a target trough level of 0.5 to 10 ng/ml, 0.5to 5 ng/ml, 0.5 to 3 ng/ml, 0.5 to 2 ng/ml, or 0.5 to 1 ng/ml.

In an embodiment a method disclosed herein comprises administering to asubject an mTOR inhibitor, e.g., an allosteric inhibitor, e.g., RAD001,at a dosage that provides a target trough level of 1 to 10 ng/ml, 1 to 5ng/ml, 1 to 3 ng/ml, or 1 to 2 ng/ml.

As used herein, the term “trough level” refers to the concentration of adrug in plasma just before the next dose, or the minimum drugconcentration between two doses.

In some embodiments, a target trough level of RAD001 is in a range ofbetween about 0.1 and 4.9 ng/ml. In an embodiment, the target troughlevel is below 3 ng/ml, e.g., is between 0.3 or less and 3 ng/ml. In anembodiment, the target trough level is below 3 ng/ml, e.g., is between0.3 or less and 1 ng/ml.

In a further aspect, the invention can utilize an mTOR inhibitor otherthan RAD001 in an amount that is associated with a target trough levelthat is bioequivalent to the specified target trough level for RAD001.In an embodiment, the target trough level for an mTOR inhibitor otherthan RAD001, is a level that gives the same level of mTOR inhibition(e.g., as measured by a method described herein, e.g., the inhibition ofP70 S6) as does a trough level of RAD001 described herein.

Pharmaceutical Compositions: mTOR Inhibitors

In one aspect, the present invention relates to pharmaceuticalcompositions comprising an mTOR inhibitor, e.g., an mTOR inhibitor asdescribed herein, formulated for use in combination with CAR cellsdescribed herein.

In some embodiments, the mTOR inhibitor is formulated for administrationin combination with an additional, e.g., as described herein.

In general, compounds of the invention will be administered intherapeutically effective amounts as described above via any of theusual and acceptable modes known in the art, either singly or incombination with one or more therapeutic agents.

The pharmaceutical formulations may be prepared using conventionaldissolution and mixing procedures. For example, the bulk drug substance(e.g., an mTOR inhibitor or stabilized form of the compound (e.g.,complex with a cyclodextrin derivative or other known complexationagent) is dissolved in a suitable solvent in the presence of one or moreof the excipients described herein. The mTOR inhibitor is typicallyformulated into pharmaceutical dosage forms to provide an easilycontrollable dosage of the drug and to give the patient an elegant andeasily handleable product.

Compounds of the invention can be administered as pharmaceuticalcompositions by any conventional route, in particular enterally, e.g.,orally, e.g., in the form of tablets or capsules, or parenterally, e.g.,in the form of injectable solutions or suspensions, topically, e.g., inthe form of lotions, gels, ointments or creams, or in a nasal orsuppository form. Where an mTOR inhibitor is administered in combinationwith (either simultaneously with or separately from) another agent asdescribed herein, in one aspect, both components can be administered bythe same route (e.g., parenterally). Alternatively, another agent may beadministered by a different route relative to the mTOR inhibitor. Forexample, an mTOR inhibitor may be administered orally and the otheragent may be administered parenterally.

Sustained Release

mTOR inhibitors, e.g., allosteric mTOR inhibitors or catalytic mTORinhibitors, disclosed herein can be provided as pharmaceuticalformulations in form of oral solid dosage forms comprising an mTORinhibitor disclosed herein, e.g., rapamycin or RAD001, which satisfyproduct stability requirements and/or have favorable pharmacokineticproperties over the immediate release (IR) tablets, such as reducedaverage plasma peak concentrations, reduced inter- and intra-patientvariability in the extent of drug absorption and in the plasma peakconcentration, reduced C_(max)/C_(min) ratio and/or reduced foodeffects. Provided pharmaceutical formulations may allow for more precisedose adjustment and/or reduce frequency of adverse events thus providingsafer treatments for patients with an mTOR inhibitor disclosed herein,e.g., rapamycin or RAD001.

In some embodiments, the present disclosure provides stable extendedrelease formulations of an mTOR inhibitor disclosed herein, e.g.,rapamycin or RAD001, which are multi-particulate systems and may havefunctional layers and coatings.

The term “extended release, multi-particulate formulation as used hereinrefers to a formulation which enables release of an mTOR inhibitordisclosed herein, e.g., rapamycin or RAD001, over an extended period oftime e.g. over at least 1, 2, 3, 4, 5 or 6 hours. The extended releaseformulation may contain matrices and coatings made of specialexcipients, e.g., as described herein, which are formulated in a manneras to make the active ingredient available over an extended period oftime following ingestion.

The term “extended release” can be interchangeably used with the terms“sustained release” (SR) or “prolonged release”. The term “extendedrelease” relates to a pharmaceutical formulation that does not releaseactive drug substance immediately after oral dosing but over an extendedin accordance with the definition in the pharmacopoeias Ph. Eur. (7^(th)edition) mongraph for tablets and capsules and USP general chapter<1151> for pharmaceutical dosage forms. The term “Immediate Release”(IR) as used herein refers to a pharmaceutical formulation whichreleases 85% of the active drug substance within less than 60 minutes inaccordance with the definition of “Guidance for Industry: “DissolutionTesting of Immediate Release Solid Oral Dosage Forms” (FDA CDER, 1997).In some embodiments, the term “immediate release” means release ofeverolismus from tablets within the time of 30 minutes, e.g., asmeasured in the dissolution assay described herein.

Stable extended release formulations of an mTOR inhibitor disclosedherein, e.g., rapamycin or RAD001, can be characterized by an in-vitrorelease profile using assays known in the art, such as a dissolutionassay as described herein: a dissolution vessel filled with 900 mLphosphate buffer pH 6.8 containing sodium dodecyl sulfate 0.2% at 37° C.and the dissolution is performed using a paddle method at 75 rpmaccording to USP by according to USP testing monograph 711, and Ph. Eur.testing monograph 2.9.3. respectively.

In some embodiments, stable extended release formulations of an mTORinhibitor disclosed herein, e.g., rapamycin or RAD001, release the mTORinhibitor in the in-vitro release assay according to following releasespecifications:

-   -   0.5 h: <45%, or <40, e.g., <30%    -   1 h: 20-80%, e.g., 30-60%    -   2 h: >50%, or >70%, e.g., >75%    -   3 h: >60%, or >65%, e.g., >85%, e.g., >90%.

In some embodiments, stable extended release formulations of an mTORinhibitor disclosed herein, e.g., rapamycin or RAD001, release 50% ofthe mTOR inhibitor not earlier than 45, 60, 75, 90, 105 min or 120 minin the in-vitro dissolution assay.

Pharmaceutical Compositions and Treatments

Pharmaceutical compositions of the present invention may comprise aCAR-expressing cell, e.g., a plurality of CAR-expressing cells, asdescribed herein, in combination with one or more pharmaceutically orphysiologically acceptable carriers, diluents or excipients. Suchcompositions may comprise buffers such as neutral buffered saline,phosphate buffered saline and the like; carbohydrates such as glucose,mannose, sucrose or dextrans, mannitol; proteins; polypeptides or aminoacids such as glycine; antioxidants; chelating agents such as EDTA orglutathione; adjuvants (e.g., aluminum hydroxide); and preservatives.Compositions of the present invention are in one aspect formulated forintravenous administration.

Pharmaceutical compositions of the present invention may be administeredin a manner appropriate to the disease to be treated (or prevented). Thequantity and frequency of administration will be determined by suchfactors as the condition of the patient, and the type and severity ofthe patient's disease, although appropriate dosages may be determined byclinical trials.

In one embodiment, the pharmaceutical composition is substantially freeof, e.g., there are no detectable levels of a contaminant, e.g.,selected from the group consisting of endotoxin, mycoplasma, replicationcompetent lentivirus (RCL), p24, VSV-G nucleic acid, HIV gag, residualanti-CD3/anti-CD28 coated beads, mouse antibodies, pooled human serum,bovine serum albumin, bovine serum, culture media components, vectorpackaging cell or plasmid components, a bacterium and a fungus. In oneembodiment, the bacterium is at least one selected from the groupconsisting of Alcaligenes faecalis, Candida albicans, Escherichia coli,Haemophilus influenza, Neisseria meningitides, Pseudomonas aeruginosa,Staphylococcus aureus, Streptococcus pneumonia, and Streptococcuspyogenes group A.

When “an immunologically effective amount,” “an anti-cancer effectiveamount,” “a cancer-inhibiting effective amount,” or “therapeutic amount”is indicated, the precise amount of the compositions of the presentinvention to be administered can be determined by a physician withconsideration of individual differences in age, weight, tumor size,extent of infection or metastasis, and condition of the patient(subject). It can generally be stated that a pharmaceutical compositioncomprising the immune effector cells described herein may beadministered at a dosage of 10⁴ to 10⁹ cells/kg body weight, in someinstances 10⁵ to 10⁶ cells/kg body weight, including all integer valueswithin those ranges. The immune effector cell compositions may also beadministered multiple times at these dosages. The cells can beadministered by using infusion techniques that are commonly known inimmunotherapy (see, e.g., Rosenberg et al., New Eng. J. of Med.319:1676, 1988).

In certain aspects, it may be desired to administer activated immuneeffector cells to a subject and then subsequently redraw blood (or havean apheresis performed), activate the cells therefrom according to thepresent invention, and reinfuse the patient with these activated andexpanded cells. This process can be carried out multiple times every fewweeks. In certain aspects, the cells can be activated from blood drawsof from 10 cc to 400 cc. In certain aspects, the cells are activatedfrom blood draws of 20 cc, 30 cc, 40 cc, 50 cc, 60 cc, 70 cc, 80 cc, 90cc, or 100 cc.

The administration of the subject compositions may be carried out in anyconvenient manner, including by aerosol inhalation, injection,ingestion, transfusion, implantation or transplantation. Thecompositions described herein may be administered to a patient transarterially, subcutaneously, intradermally, intratumorally, intranodally,intramedullary, intramuscularly, by intravenous (i.v.) injection, orintraperitoneally. In one aspect, the T cell compositions of the presentinvention are administered to a patient by intradermal or subcutaneousinjection. In one aspect, the immune effector cell compositions of thepresent invention are administered by i.v. injection. The compositionsof immune effector cells may be injected directly into a tumor, lymphnode, or site of infection.

In a particular exemplary aspect, subjects may undergo leukapheresis,wherein leukocytes are collected, enriched, or depleted ex vivo toselect and/or isolate the cells of interest, e.g., T cells. These T cellisolates may be expanded by methods known in the art and treated suchthat one or more CAR constructs of the invention may be introduced,thereby creating a CAR T cell of the invention. Subjects in need thereofmay subsequently undergo standard treatment with high dose chemotherapyfollowed by peripheral blood stem cell transplantation. In certainaspects, following or concurrent with the transplant, subjects receivean infusion of the expanded CAR T cells of the present invention. In anadditional aspect, expanded cells are administered before or followingsurgery.

The dosage of the above treatments to be administered to a patient willvary with the precise nature of the condition being treated and therecipient of the treatment. The scaling of dosages for humanadministration can be performed according to art-accepted practices. Thedose for CAMPATH, for example, will generally be in the range 1 to about100 mg for an adult patient, usually administered daily for a periodbetween 1 and 30 days. The preferred daily dose is 1 to 10 mg per dayalthough in some instances larger doses of up to 40 mg per day may beused (described in U.S. Pat. No. 6,120,766).

In one embodiment, the CAR is introduced into immune effector cells,e.g., using in vitro transcription, and the subject (e.g., human)receives an initial administration of CAR-expressingcells of theinvention, and one or more subsequent administrations of theCAR-expressing cells of the invention, wherein the one or moresubsequent administrations are administered less than 15 days, e.g., 14,13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, or 2 days after the previousadministration. In one embodiment, more than one administration of theCAR-expressing cells of the invention are administered to the subject(e.g., human) per week, e.g., 2, 3, or 4 administrations of theCAR-expressing cells of the invention are administered per week. In oneembodiment, the subject (e.g., human subject) receives more than oneadministration of the CAR-expressing cells per week (e.g., 2, 3 or 4administrations per week) (also referred to herein as a cycle), followedby a week of no CAR-expressing cells administration, and then one ormore additional administration of the CAR-expressing cells (e.g., morethan one administration of the CAR-expressing cells per week) isadministered to the subject. In another embodiment, the subject (e.g.,human subject) receives more than one cycle of CAR-expressing cells, andthe time between each cycle is less than 10, 9, 8, 7, 6, 5, 4, or 3days. In one embodiment, the CAR-expressing cells are administered everyother day for 3 administrations per week. In one embodiment, theCAR-expressing cells of the invention are administered for at least two,three, four, five, six, seven, eight or more weeks.

In one aspect, mesothelin CAR-expressing cells are generated usinglentiviral viral vectors, such as lentivirus. CAR-expressing cellsgenerated that way will have stable CAR expression.

In one aspect, the CAR-expressing cellss transiently express CAR vectorsfor 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 days after transduction.Transient expression of CARs can be effected by RNA CAR vector delivery.In one aspect, the CAR RNA is transduced into the T cell byelectroporation.

In one embodiment, the dose and/or dosing schedule is one provided inFIG. 6.

A potential issue that can arise in patients being treated usingtransiently expressing CAR-expressing cells (particularly with murinescFv bearing CAR-expressing cells) is anaphylaxis after multipletreatments.

Without being bound by this theory, it is believed that such ananaphylactic response might be caused by a patient developing humoralanti-CAR response, i.e., anti-CAR antibodies having an anti-IgE isotype.It is thought that a patient's antibody producing cells undergo a classswitch from IgG isotype (that does not cause anaphylaxis) to IgE isotypewhen there is a ten to fourteen day break in exposure to antigen.

If a patient is at high risk of generating an anti-CAR antibody responseduring the course of transient CAR therapy (such as those generated byRNA transductions), CAR-expressing cell infusion breaks should not lastmore than ten to fourteen days.

Using CARs with human (instead of murine) scFvs can reduce thelikelihood and intensity of a patient having an anti-CAR response.

TABLE 2Amino Acid Sequences of Human scFvs and CARs (bold underline is theleader sequence and bold without underline is a linker sequence). In the case of the scFvs, theremaining amino acids are the heavy chain variable region and light chain variable regions,with each of the HC CDRs (HC CDR1, HC CDR2, HC CDR3) and LC CDRs (LC CDR1, LCCDR2, LC CDR3) underlined). In the case of the CARs, the further remaining amino acids arethe remaining amino acids of the CARs.) SEQ ID NO: DescriptionAmino Acid Sequence 39 M1 (ScFv QVQLQQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQdomain) APGQGLEWMGRINPNSGGTNYAQKFQGRVTMTRDTSISTAYMELSRLRSEDTAVYYCARGRYYGMDVWGQGTMVTVSSGGGGSGGGGSGGGGSGGGGSEIVLTQSPATLSLSPGERATISCRASQSVSSNFAWYQQRPGQAPRLLIYDASNRATGIPPRFSGSGSGTDFTLTISSLEPEDFAAYYCHQRSNWLYTFGQGTKVDIK 63 M1 (full) MALPVTALLLPLALLLHAARPQVQLQQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQ >ZA53-APGQGLEWMGRINPNSGGTNYAQKFQGRVTMTRDTSISTAYMELSRLRSEDTAVYYCARG 27BC (M1RYYGMDVWGQGTMVTVSSGGGGSGGGGSGGGGSGGGGSEIVLTQSPATLSLSPGERATIS ZA53-27BCCRASQSVSSNFAWYQQRPGQAPRLLIYDASNRATGIPPRFSGSGSGTDFTLTISSLEPED R001-A11FAAYYCHQRSNWLYTFGQGTKVDIKTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAV 126161)HTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDAL HMQALPPR 40M2 (ScFv QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQ domain)APGQGLEWMGWINPNSGGTNYAQKFQGRVTMTRDTSISTAYMELSRLRSDDTAVYYCARDLRRTVVTPRAYYGMDVWGQGTTVTVSSGGGGSGGGGSGGGGSGGGGSDIQLTQSPSTLSASVGDRVTITCQASQDISNSLNWYQQKAGKAPKLLIYDASTLETGVPSRFSGSGSGTDFSFTISSLQPEDIATYYCQQHDNLPLTFGQGTKVEIK 64 M2 (full) MALPVTALLLPLALLLHAARPQVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQ >FA56-APGQGLEWMGWINPNSGGTNYAQKFQGRVTMTRDTSISTAYMELSRLRSDDTAVYYCARD 26RC (M2LRRTVVTPRAYYGMDVWGQGTTVTVSSGGGGSGGGGSGGGGSGGGGSDIQLTQSPSTLSA FA56-26RCSVGDRVTITCQASQDISNSLNWYQQKAGKAPKLLIYDASTLETGVPSRFSGSGSGTDFSF R001-A10TISSLQPEDIATYYCQQHDNLPLTFGQGTKVEIKTTTPAPRPPTPAPTIASQPLSLRPEA 126162)CRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR 41 M3 (ScFv QVQLVQSGAEVKKPGAPVKVSCKASGYTFTGYYMHWVRQdomain) APGQGLEWMGWINPNSGGTNYAQKFQGRVTMTRDTSISTAYMELSRLRSDDTAVYYCARGEWDGSYYYDYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSDIVLTQTPSSLSASVGDRVTITCRASQSINTYLNWYQHKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSFSPLTFGGGTKLEIK 65 M3 MALPVTALLLPLALLLHAARPQVQLVQSGAEVKKPGAPVKVSCKASGYTFTGYYMHWVRQ >VA58-APGQGLEWMGWINPNSGGTNYAQKFQGRVTMTRDTSISTAYMELSRLRSDDTAVYYCARG 21LC (M3EWDGSYYYDYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSDIVLTQTPSSLSASVGDRV VA58-21LCTITCRASQSINTYLNWYQHKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQ R001-A1PEDFATYYCQQSFSPLTFGGGTKLEIKTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGG 126163)AVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYD ALHMQALPPR42 M4 (ScFv QVQLVESGGGLVQPGGSLRLSCAASGFTFSSYWMHWVRQ domain)VPGKGLVWVSRINTDGSTTTYADSVEGRFTISRDNAKNTLYLQMNSLRDDDTAVYYCVGGHWAVWGQGTTVTVSSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSTLSASVGDRVTITCRASQSISDRLAWYQQKPGKAPKLLIYKASSLESGVPSRFSGSGSGTEFTLTISSLQPDDFAVYYCQQYGHLPMYTFGQGTKVEIK 66 M4 MALPVTALLLPLALLLHAARPQVQLVESGGGLVQPGGSLRLSCAASGFTFSSYWMHWVRQ >DP37-VPGKGLVWVSRINTDGSTTTYADSVEGRFTISRDNAKNTLYLQMNSLRDDDTAVYYCVGG 07IC (M4HWAVWGQGTTVTVSSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSTLSASVGDRVTITCRA DP37-07ICSQSISDRLAWYQQKPGKAPKLLIYKASSLESGVPSRFSGSGSGTEFTLTISSLQPDDFAV R001-C6YYCQQYGHLPMYTFGQGTKVEIKTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHT 126164)RGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHM QALPPR 43M5 (ScFv QVQLVQSGAEVEKPGASVKVSCKASGYTFTDYYMHWVRQ domain)APGQGLEWMGWINPNSGGTNYAQKFQGRVTMTRDTSISTAYMELSRLRSDDTAVYYCASGWDFDYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSDIVMTQSPSSLSASVGDRVTITCRASQSIRYYLSWYQQKPGKAPKLLIYTASILQNGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCLQTYTTPDFGPGTKVEIK 67 M5 MALPVTALLLPLALLLHAARPQVQLVQSGAEVEKPGASVKVSCKASGYTFTDYYMHWVRQ >XP31-APGQGLEWMGWINPNSGGTNYAQKFQGRVTMTRDTSISTAYMELSRLRSDDTAVYYCASG 20LC (M5WDFDYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSDIVMTQSPSSLSASVGDRVTITCR XP31-20LCASQSIRYYLSWYQQKPGKAPKLLIYTASILQNGVPSRFSGSGSGTDFTLTISSLQPEDFA R001-B4TYYCLQTYTTPDFGPGTKVEIKTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTR 126165)GLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQ ALPPR 44M6 (ScFv QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYMHWVRQ domain)APGQGLEWMGIINPSGGSTSYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARYRLIAVAGDYYYYGMDVWGQGTMVTVSSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSSVSASVGDRVTITCRASQGVGRWLAWYQQKPGTAPKLLIYAASTLQSGVPSRFSGSGSGTDFTLTINNLQPEDFATYYCQQANSFPLTFGGGTRLEIK 68 M6 MALPVTALLLPLALLLHAARPQVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYMHWVRQ >FE10-APGQGLEWMGIINPSGGSTSYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARY 06ID (M6RLIAVAGDYYYYGMDVWGQGTMVTVSSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSSVSA 46FE10-SVGDRVTITCRASQGVGRWLAWYQQKPGTAPKLLIYAASTLQSGVPSRFSGSGSGTDFTL 06IDTINNLQPEDFATYYCQQANSFPLTFGGGTRLEIKTTTPAPRPPTPAPTIASQPLSLRPEA R001-A4CRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMR 126166)PVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR 45 M7 (ScFv QVQLVQSGGGVVQPGRSLRLSCAASGFTFSSYAMHWVRQdomain) APGKGLEWVAVISYDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARWKVSSSSPAFDYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSEIVLTQSPATLSLSPGERAILSCRASQSVYTKYLGWYQQKPGQAPRLLIYDASTRATGIPDRFSGSGSGTDFTLTINRLEPEDFAVYYCQHYGGSPLITFGQGTRLEIK 69 M7 MALPVTALLLPLALLLHAARPQVQLVQSGGGVVQPGRSLRLSCAASGFTFSSYAMHWVRQ >VE12-APGKGLEWVAVISYDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARW 01CD (M7KVSSSSPAFDYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSEIVLTQSPATLSLSPGER VE12-01CDAILSCRASQSVYTKYLGWYQQKPGQAPRLLIYDASTRATGIPDRFSGSGSGTDFTLTINR R001-A5LEPEDFAVYYCQHYGGSPLITFGQGTRLEIKTTTPAPRPPTPAPTIASQPLSLRPEACRP 126167)AAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR 46 M8 (ScFv QVQLQQSGAEVKKPGASVKVSCKTSGYPFTGYSLHWVRQdomain) APGQGLEWMGWINPNSGGTNYAQKFQGRVTMTRDTSISTAYMELSRLRSDDTAVYYCARDHYGGNSLFYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSDIQLTQSPSSISASVGDTVSITCRASQDSGTWLAWYQQKPGKAPNLLMYDASTLEDGVPSRFSGSASGTEFTLTVNRLQPEDSATYYCQQYNSYPLTFGGGTKVDIK 70 M8 MALPVTALLLPLALLLHAARPQVQLQQSGAEVKKPGASVKVSCKTSGYPFTGYSLHWVRQ >LE13-APGQGLEWMGWINPNSGGTNYAQKFQGRVTMTRDTSISTAYMELSRLRSDDTAVYYCARD 05XD (M8HYGGNSLFYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSDIQLTQSPSSISASVGDTVS LE13-05XDITCRASQDSGTWLAWYQQKPGKAPNLLMYDASTLEDGVPSRFSGSASGTEFTLTVNRLQP R001-E5EDSATYYCQQYNSYPLTFGGGTKVDIKTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGG 126168)AVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYD ALHMQALPPR47 M9 (ScFv QVQLVQSGAEVKKPGASVEVSCKASGYTFTSYYMHWVRQ domain)APGQGLEWMGIINPSGGSTGYAQKFQGRVTMTRDTSTSTVHMELSSLRSEDTAVYYCARGGYSSSSDAFDIWGQGTMVTVSSGGGGSGGGGSGGGGSGGGGSDIQMTQSPPSLSASVGDRVTITCRASQDISSALAWYQQKPGTPPKLLIYDASSLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQFSSYPLTFGGGTRLEIK 71 M9 MALPVTALLLPLALLLHAARPQVQLVQSGAEVKKPGASVEVSCKASGYTFTSYYMHWVRQ >BE15-APGQGLEWMGIINPSGGSTGYAQKFQGRVTMTRDTSTSTVHMELSSLRSEDTAVYYCARG 00SD (M9GYSSSSDAFDIWGQGTMVTVSSGGGGSGGGGSGGGGSGGGGSDIQMTQSPPSLSASVGDR BE15-00SDVTITCRASQDISSALAWYQQKPGTPPKLLIYDASSLESGVPSRFSGSGSGTDFTLTISSL R001-A3QPEDFATYYCQQFSSYPLTFGGGTRLEIKTTTPAPRPPTPAPTIASQPLSLRPEACRPAA 126169)GGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR 48 M10 (ScFv QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYGISWVRQdomain) APGQGLEWMGWISAYNGNTNYAQKLQGRVTMTTDTSTSTAYMELRSLRSDDTAVYYCARVAGGIYYYYGMDVWGQGTTITVSSGGGGSGGGGSGGGGSGGGGSDIVMTQTPDSLAVSLGERATISCKSSHSVLYNRNNKNYLAWYQQKPGQPPKLLFYWASTRKSGVPDRFSGSGSGTDFTLTISSLQPEDFATYFCQQTQTFPLTFGQGTRLEIN 72 M10 MALPVTALLLPLALLLHAARPQVQLVQSGAEVKKPGASVKVSCKASGYTFTSYGISWVRQ >RE16-APGQGLEWMGWISAYNGNTNYAQKLQGRVTMTTDTSTSTAYMELRSLRSDDTAVYYCARV 05MD (M10AGGIYYYYGMDVWGQGTTITVSSGGGGSGGGGSGGGGSGGGGSDIVMTQTPDSLAVSLGE RE16-05MDRATISCKSSHSVLYNRNNKNYLAWYQQKPGQPPKLLFYWASTRKSGVPDRFSGSGSGTDF R001-D10TLTISSLQPEDFATYFCQQTQTFPLTFGQGTRLEINTTTPAPRPPTPAPTIASQPLSLRP 126170)EACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR 49 M11 (ScFv QVQLQQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQdomain) APGQGLEWMGWINPNSGGTNYAQNFQGRVTMTRDTSISTAYMELRRLRSDDTAVYYCASGWDFDYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSDIRMTQSPSSLSASVGDRVTITCRASQSIRYYLSWYQQKPGKAPKLLIYTASILQNGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCLQTYTTPDFGPGTKVEIK 73 M11 MALPVTALLLPLALLLHAARPQVQLQQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQ >NE10-APGQGLEWMGWINPNSGGTNYAQNFQGRVTMTRDTSISTAYMELRRLRSDDTAVYYCASG 19WD (M11WDFDYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSDIRMTQSPSSLSASVGDRVTITCR NE10-19WDASQSIRYYLSWYQQKPGKAPKLLIYTASILQNGVPSRFSGSGSGTDFTLTISSLQPEDFA R001-G2TYYCLQTYTTPDFGPGTKVEIKTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTR 126171)GLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQ ALPPR 50M12 (ScFv QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQ domain)APGQGLEWMGRINPNSGGTNYAQKFQGRVTMTTDTSTSTAYMELRSLRSDDTAVYYCARTTTSYAFDIWGQGTMVTVSSGGGGSGGGGSGGGGSGGGGSDIQLTQSPSTLSASVGDRVTITCRASQSISTWLAWYQQKPGKAPNLLIYKASTLESGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQQYNTYSPYTFGQGTKLEIK 74 M12 MALPVTALLLPLALLLHAARPQVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQ >DE12-APGQGLEWMGRINPNSGGTNYAQKFQGRVTMTTDTSTSTAYMELRSLRSDDTAVYYCART 14RD (M12TTSYAFDIWGQGTMVTVSSGGGGSGGGGSGGGGSGGGGSDIQLTQSPSTLSASVGDRVTI DE12-14RDTCRASQSISTWLAWYQQKPGKAPNLLIYKASTLESGVPSRFSGSGSGTEFTLTISSLQPD R001-G9DFATYYCQQYNTYSPYTFGQGTKLEIKTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGG 126172)AVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYD ALHMQALPPR51 M13 (ScFv QVQLVQSGGGLVKPGGSLRLSCEASGFIFSDYYMGWIRQ domain)APGKGLEWVSYIGRSGSSMYYADSVKGRFTFSRDNAKNSLYLQMNSLRAEDTAVYYCAASPVVAATEDFQHWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSDIVMTQTPATLSLSPGERATLSCRASQSVTSNYLAWYQQKPGQAPRLLLFGASTRATGIPDRFSGSGSGTDFTLTINRLEPEDFAMYYCQQYGSAPVTFGQGTKLEIK 75 M13 MALPVTALLLPLALLLHAARPQVQLVQSGGGLVKPGGSLRLSCEASGFIFSDYYMGWIRQ >TE13-APGKGLEWVSYIGRSGSSMYYADSVKGRFTFSRDNAKNSLYLQMNSLRAEDTAVYYCAAS 19LD (M13PVVAATEDFQHWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSDIVMTQTPATLSLSPGER TE13-19LDATLSCRASQSVTSNYLAWYQQKPGQAPRLLLFGASTRATGIPDRFSGSGSGTDFTLTINR R002-C3LEPEDFAMYYCQQYGSAPVTFGQGTKLEIKTTTPAPRPPTPAPTIASQPLSLRPEACRPA 126173)AGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR 52 M14 (ScFv QVQLVQSGAEVRAPGASVKISCKASGFTFRGYYIHWVRQdomain) APGQGLEWMGIINPSGGSRAYAQKFQGRVTMTRDTSTSTVYMELSSLRSDDTAMYYCARTASCGGDCYYLDYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSDIQMTQSPPTLSASVGDRVTITCRASENVNIWLAWYQQKPGKAPKLLIYKSSSLASGVPSRFSGSGSGAEFTLTISSLQPDDFATYYCQQYQSYPLTEGGGTKVDIK 76 M14 MALPVTALLLPLALLLHAARPQVQLVQSGAEVRAPGASVKISCKASGFTFRGYYIHWVRQ >BS83-APGQGLEWMGIINPSGGSRAYAQKFQGRVTMTRDTSTSTVYMELSSLRSDDTAMYYCART 95ID (M14ASCGGDCYYLDYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSDIQMTQSPPTLSASVGD BS83-95IDRVTITCRASENVNIWLAWYQQKPGKAPKLLIYKSSSLASGVPSRFSGSGSGAEFTLTISS R001-E8LQPDDFATYYCQQYQSYPLTFGGGTKVDIKTTTPAPRPPTPAPTIASQPLSLRPEACRPA 126174)AGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR 53 M15 (ScFv QVQLVQSGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQdomain) APGKGLEWVSGISWNSGSIGYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAKDGSSSWSWGYFDYWGQGTLVTVSSGGGGSGGGGSGGGGSSSELTQDPAVSVALGQTVRTTCQGDALRSYYASWYQQKPGQAPMLVIYGKNNRPSGIPDRFSGSDSGDTASLTITGAQAEDEADYYCNSRDSSGYPVFGTGTKVTVL 77 M15 MALPVTALLLPLALLLHAARPQVQLVQSGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQ >HS86-APGKGLEWVSGISWNSGSIGYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAKD 94XD (M15GSSSWSWGYFDYWGQGTLVTVSSGGGGSGGGGSGGGGSSSELTQDPAVSVALGQTVRTTC HS86-94XDQGDALRSYYASWYQQKPGQAPMLVIYGKNNRPSGIPDRFSGSDSGDTASLTITGAQAEDE NTADYYCNSRDSSGYPVFGTGTKVTVLTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAV 127553)HTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDAL HMQALPPR 54M16 (ScFv EVQLVESGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQ domain)APGKGLEWVSGISWNSGSTGYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTALYYCAKDSSSWYGGGSAFDIWGQGTMVTVSSGGGGSGGGGSGGGGSSSELTQEPAVSVALGQTVRITCQGDSLRSYYASWYQQKPGQAPVLVIFGRSRRPSGIPDRFSGSSSGNTASLIITGAQAEDEADYYCNSRDNTANHYVFGTGTKLTVL 78 M16 MALPVTALLLPLALLLHAARPEVQLVESGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQ >XS87-APGKGLEWVSGISWNSGSTGYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTALYYCAKD 99RD (M16SSSWYGGGSAFDIWGQGTMVTVSSGGGGSGGGGSGGGGSSSELTQEPAVSVALGQTVRIT XS87-99RDCQGDSLRSYYASWYQQKPGQAPVLVIFGRSRRPSGIPDRFSGSSSGNTASLIITGAQAED NTEADYYCNSRDNTANHYVFGTGTKLTVLTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGG 127554)AVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYD ALHMQALPPR55 M17 (ScFv EVQLVESGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQ domain)APGKGLEWVSGISWNSGSTGYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTALYYCAKDSSSWYGGGSAFDIWGQGTMVTVSSGGGGSGGGGSGGGGSSSELTQDPAVSVALGQTVRITCQGDSLRSYYASWYQQKPGQAPVLVIYGKNNRPSGIPDRFSGSSSGNTASLTITGAQAEDEADYYCNSRGSSGNHYVFGTGTKVTVL 79 M17 MALPVTALLLPLALLLHAARPEVQLVESGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQ >NX89-APGKGLEWVSGISWNSGSTGYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTALYYCAKD 94MD (M17SSSWYGGGSAFDIWGQGTMVTVSSGGGGSGGGGSGGGGSSSELTQDPAVSVALGQTVRIT NS89-94MDCQGDSLRSYYASWYQQKPGQAPVLVIYGKNNRPSGIPDRFSGSSSGNTASLTITGAQAED NTEADYYCNSRGSSGNHYVFGTGTKVTVLTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGG 127555)AVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYD ALHMQALPPR56 M18 (ScFv QVQLVQSGGGLVQPGGSLRLSCAASGFTFSSYWMHWVRQ domain)APGKGLVWVSRINSDGSSTSYADSVKGRFTISRDNAKNTLYLQMNSLRAEDTAVYYCVRTGWVGSYYYYMDVWGKGTTVTVSSGGGGSGGGGSGGGGSGGGGSEIVLTQSPGTLSLSPGERATLSCRASQSVSSNYLAWYQQKPGQPPRLLIYDVSTRATGIPARFSGGGSGTDFTLTISSLEPEDFAVYYCQQRSNWPPWTFGQGTKVEIK 80 M18 MALPVTALLLPLALLLHAARPQVQLVQSGGGLVQPGGSLRLSCAASGFTFSSYWMHWVRQ >DS90-APGKGLVWVSRINSDGSSTSYADSVKGRFTISRDNAKNTLYLQMNSLRAEDTAVYYCVRT 09HD (M18GWVGSYYYYMDVWGKGTTVTVSSGGGGSGGGGSGGGGSGGGGSEIVLTQSPGTLSLSPGE DS90-09HDRATLSCRASQSVSSNYLAWYQQKPGQPPRLLIYDVSTRATGIPARFSGGGSGTDFTLTIS R003-A05SLEPEDFAVYYCQQRSNWPPWTFGQGTKVEIKTTTPAPRPPTPAPTIASQPLSLRPEACR 127556)PAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR 57 M19 (ScFv QVQLVQSGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQdomain) APGKGLEWVAVISYDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKGYSRYYYYGMDVWGQGTTVTVSSGGGGSGGGGSGGGGSGGGGSEIVMTQSPATLSLSPGERAILSCRASQSVYTKYLGWYQQKPGQAPRLLIYDASTRATGIPDRFSGSGSGTDFTLTINRLEPEDFAVYYCQHYGGSPLITFGQGTKVDIK 81 M19 MALPVTALLLPLALLLHAARPQVQLVQSGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQ >TS92-APGKGLEWVAVISYDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKG 04BD (M19YSRYYYYGMDVWGQGTTVTVSSGGGGSGGGGSGGGGSGGGGSEIVMTQSPATLSLSPGER TS92-04BDAILSCRASQSVYTKYLGWYQQKPGQAPRLLIYDASTRATGIPDRFSGSGSGTDFTLTINR R003-006LEPEDFAVYYCQHYGGSPLITFGQGTKVDIKTTTPAPRPPTPAPTIASQPLSLRPEACRP 127557)AAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR 58 M20 (ScFv QVQLVQSGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQdomain) APGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKREAAAGHDWYFDLWGRGTLVTVSSGGGGSGGGGSGGGGSGGGGSDIRVTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSIPLTFGQGTKVEIK 82 M20 MALPVTALLLPLALLLHAARPQVQLVQSGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQ >JS93-APGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKR 08WD (M20EAAAGHDWYFDLWGRGTLVTVSSGGGGSGGGGSGGGGSGGGGSDIRVTQSPSSLSASVGD JS93-08WDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISS R003-E07LQPEDFATYYCQQSYSIPLTFGQGTKVEIKTTTPAPRPPTPAPTIASQPLSLRPEACRPA 127558)AGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR 275 Ss1 (scFvQVQLQQSGPELEKPGASVKISCKASGYSFTGYTMNWVKQSHGKSLEWIGLITPYNGASS domain)YNQKFRGKATLTVDKSSSTAYMDLLSLTSEDSAVYFCARGGYDGRGFDYWGQGTTVTVSSGGGGSGGGGSGGGGSDIELTQSPAIMSASPGEKVTMTCSASSSVSYMHWYQQKSGTSPKRWIYDTSKLASGVPGRFSGSGSGNSYSLTISSVEAEDDATYYCQQWSGYPLTFGAGTK LET 391Ss1 (full) MALPVTALLLPLALLLHAARPQVQLQQSGPELEKPGASVKISCKASGYSFTGYTMNWVKQSHGKSLEWIGLITPYNGASSYNQKFRGKATLTVDKSSSTAYMDLLSLTSEDSAVYFCARGGYDGRGFDYWGQGTTVTVSSGGGGSGGGGSGGGGSDIELTQSPAIMSASPGEKVTMTCSASSSVSYMHWYQQKSGTSPKRWIYDTSKLASGVPGRFSGSGSGNSYSLTISSVEAEDDATYYCQQWSGYPLTFGAGTKLEITTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPA

TABLE 3Nucleic Acid Sequences encoding CAR molecules (underlined is the leader sequence)SEQ ID NO: Desc. Nucleic Acid Sequence 87 M1CAAGTCCAACTGCAGCAGTCAGGAGCGGAAGTGAAGAAACCAGGAGCGTCAGTCAAAGTGTCGTGCAAGGCTAGCGGCTA(ScFv CACCTTCACCGGCTACTA domain)CATGCACTGGGTTCGACAGGCTCCAGGGCAGGGTCTGGAGTGGATGGGCCGCATCAACCCGAATTCCGGTGGGACTAACT >ZA53-ACGCCCAGAAGTTCCAGGGAAGAGTGACCATGACTAGGGACACGTCGATCAGCACTGCGTACATGGAACTGAGCCGCCTG27BCCGGTCCGAGGATACTGCCGTCTACTACTGCGCACGCGGAAGGTACTATGGAATGGACGTGTGGGGCCAAGGGACTATGGT(M1)GACTGTGAGCTCGGGAGGGGGAGGCTCCGGTGGCGGGGGATCAGGAGGAGGAGGATCAGGGGGAGGAGGTTCCGAAATTGTCCTCACCCAGAGCCCGGCAACCCTCTCACTTTCCCCGGGAGAGCGCGCAACCATCTCTTGCCGGGCTAGCCAATCCGTGTCGTCCAATTTCGCCTGGTACCAGCAACGGCCGGGACAAGCCCCTAGACTCCTGATCTACGACGCCAGCAACAGAGCGACTGGAATTCCTCCACGCTTTTCGGGATCAGGCTCCGGTACCGACTTCACCCTGACTATCTCGTCGCTCGAACCCGAGGATTTCGCCGCCTACTACTGTCATCAGCGGTCGAACTGGTTGTATACGTTTGGCCAGGGCACCAAGGTGGATATCAAG111 M1ATGGCCCTCCCTGTCACCGCCCTGCTGCTTCCGCTGGCTCTTCTGCTCCACGCCGCTCGGCCCCAAGTCCAACTGCAGCA(Full) G >ZA53-TCAGGAGCGGAAGTGAAGAAACCAGGAGCGTCAGTCAAAGTGTCGTGCAAGGCTAGCGGCTACACCTTCACCGGCTACTA27BCCATGCACTGGGTTCGACAGGCTCCAGGGCAGGGTCTGGAGTGGATGGGCCGCATCAACCCGAATTCCGGTGGGACTAACT(M1)ACGCCCAGAAGTTCCAGGGAAGAGTGACCATGACTAGGGACACGTCGATCAGCACTGCGTACATGGAACTGAGCCGCCTGCGGTCCGAGGATACTGCCGTCTACTACTGCGCACGCGGAAGGTACTATGGAATGGACGTGTGGGGCCAAGGGACTATGGTGACTGTGAGCTCGGGAGGGGGAGGCTCCGGTGGCGGGGGATCAGGAGGAGGAGGATCAGGGGGAGGAGGTTCCGAAATTGTCCTCACCCAGAGCCCGGCAACCCTCTCACTTTCCCCGGGAGAGCGCGCAACCATCTCTTGCCGGGCTAGCCAATCCGTGTCGTCCAATTTCGCCTGGTACCAGCAACGGCCGGGACAAGCCCCTAGACTCCTGATCTACGACGCCAGCAACAGAGCGACTGGAATTCCTCCACGCTTTTCGGGATCAGGCTCCGGTACCGACTTCACCCTGACTATCTCGTCGCTCGAACCCGAGGATTTCGCCGCCTACTACTGTCATCAGCGGTCGAACTGGTTGTATACGTTTGGCCAGGGCACCAAGGTGGATATCAAGACCACTACCCCAGCACCGAGGCCACCCACCCCGGCTCCTACCATCGCCTCCCAGCCTCTGTCCCTGCGTCCGGAGGCATGTAGACCCGCAGCTGGTGGGGCCGTGCATACCCGGGGTCTTGACTTCGCCTGCGATATCTACATTTGGGCCCCTCTGGCTGGTACTTGCGGGGTCCTGCTGCTTTCACTCGTGATCACTCTTTACTGTAAGCGCGGTCGGAAGAAGCTGCTGTACATCTTTAAGCAACCCTTCATGAGGCCTGTGCAGACTACTCAAGAGGAGGACGGCTGTTCATGCCGGTTCCCAGAGGAGGAGGAAGGCGGCTGCGAACTGCGCGTGAAATTCAGCCGCAGCGCAGATGCTCCAGCCTACAAGCAGGGGCAGAACCAGCTCTACAACGAACTCAATCTTGGTCGGAGAGAGGAGTACGACGTGCTGGACAAGCGGAGAGGACGGGACCCAGAAATGGGCGGGAAGCCGCGCAGAAAGAATCCCCAAGAGGGCCTGTACAACGAGCTCCAAAAGGATAAGATGGCAGAAGCCTATAGCGAGATTGGTATGAAAGGGGAACGCAGAAGAGGCAAAGGCCACGACGGACTGTACCAGGGACTCAGCACCGCCACCAAGGACACCTATGACGCTCTTCACATGCAGGCCCTGCCGCCTCGG 88 M2CAAGTCCAACTCGTCCAGTCAGGAGCAGAAGTCAAGAAACCAGGTGCTAGCGTGAAAGTGTCGTGCAAGGCGTCGGGATA(ScFv CACTTTCACCGGATACTAC domain)ATGCACTGGGTCCGCCAGGCCCCCGGACAAGGACTGGAATGGATGGGCTGGATCAACCCGAATAGCGGGGGAACTAATTA >FA56-CGCCCAGAAGTTTCAGGGACGAGTGACCATGACCCGCGATACCTCTATCTCGACCGCCTACATGGAGCTCTCCAGACTGC26RCGCTCCGACGATACTGCAGTGTACTACTGCGCCCGGGACCTGAGGCGGACTGTGGTTACTCCTCGCGCCTATTATGGCATG(M2)GACGTGTGGGGCCAAGGAACTACTGTGACTGTGAGCTCGGGAGGCGGTGGGTCAGGCGGAGGAGGGTCGGGCGGTGGTGGCTCGGGAGGGGGAGGAAGCGACATTCAACTTACGCAGAGCCCGTCAACCCTGTCAGCGTCAGTGGGAGATCGGGTGACCATCACGTGTCAGGCCAGCCAGGATATCTCCAACTCGCTCAACTGGTACCAGCAAAAGGCGGGTAAAGCTCCGAAGCTGCTGATCTACGACGCTTCCACCCTCGAGACTGGAGTCCCATCCAGATTTTCCGGGTCAGGAAGCGGCACCGATTTCTCCTTCACCATTTCGTCCTTGCAACCGGAGGACATCGCAACCTACTACTGCCAGCAGCATGACAACTTGCCTCTGACGTTCGGGCAGGGCACCAAGGTGGAAATCAAG 112 M2ATGGCCCTCCCTGTCACCGCCCTGCTGCTTCCGCTGGCTCTTCTGCTCCACGCCGCTCGGCCCCAAGTCCAACTCGTCCA(Full)GTCAGGAGCAGAAGTCAAGAAACCAGGTGCTAGCGTGAAAGTGTCGTGCAAGGCGTCGGGATACACTTTCACCGGATACT >FA56-AC 26RCATGCACTGGGTCCGCCAGGCCCCCGGACAAGGACTGGAATGGATGGGCTGGATCAACCCGAATAGCGGGGGAACTAATTA(M2)CGCCCAGAAGTTTCAGGGACGAGTGACCATGACCCGCGATACCTCTATCTCGACCGCCTACATGGAGCTCTCCAGACTGCGCTCCGACGATACTGCAGTGTACTACTGCGCCCGGGACCTGAGGCGGACTGTGGTTACTCCTCGCGCCTATTATGGCATGGACGTGTGGGGCCAAGGAACTACTGTGACTGTGAGCTCGGGAGGCGGTGGGTCAGGCGGAGGAGGGTCGGGCGGTGGTGGCTCGGGAGGGGGAGGAAGCGACATTCAACTTACGCAGAGCCCGTCAACCCTGTCAGCGTCAGTGGGAGATCGGGTGACCATCACGTGTCAGGCCAGCCAGGATATCTCCAACTCGCTCAACTGGTACCAGCAAAAGGCGGGTAAAGCTCCGAAGCTGCTGATCTACGACGCTTCCACCCTCGAGACTGGAGTCCCATCCAGATTTTCCGGGTCAGGAAGCGGCACCGATTTCTCCTTCACCATTTCGTCCTTGCAACCGGAGGACATCGCAACCTACTACTGCCAGCAGCATGACAACTTGCCTCTGACGTTCGGGCAGGGCACCAAGGTGGAAATCAAGACCACTACCCCAGCACCGAGGCCACCCACCCCGGCTCCTACCATCGCCTCCCAGCCTCTGTCCCTGCGTCCGGAGGCATGTAGACCCGCAGCTGGTGGGGCCGTGCATACCCGGGGTCTTGACTTCGCCTGCGATATCTACATTTGGGCCCCTCTGGCTGGTACTTGCGGGGTCCTGCTGCTTTCACTCGTGATCACTCTTTACTGTAAGCGCGGTCGGAAGAAGCTGCTGTACATCTTTAAGCAACCCTTCATGAGGCCTGTGCAGACTACTCAAGAGGAGGACGGCTGTTCATGCCGGTTCCCAGAGGAGGAGGAAGGCGGCTGCGAACTGCGCGTGAAATTCAGCCGCAGCGCAGATGCTCCAGCCTACAAGCAGGGGCAGAACCAGCTCTACAACGAACTCAATCTTGGTCGGAGAGAGGAGTACGACGTGCTGGACAAGCGGAGAGGACGGGACCCAGAAATGGGCGGGAAGCCGCGCAGAAAGAATCCCCAAGAGGGCCTGTACAACGAGCTCCAAAAGGATAAGATGGCAGAAGCCTATAGCGAGATTGGTATGAAAGGGGAACGCAGAAGAGGCAAAGGCCACGACGGACTGTACCAGGGACTCAGCACCGCCACCAAGGACACCTATGACGCTCTTCACATGCAGGCCCTGCCGCCTCGG 89 M3CAAGTCCAACTCGTCCAA (ScFvTCAGGAGCGGAAGTCAAAAAGCCCGGAGCTCCAGTGAAAGTGTCATGCAAGGCCTCCGGCTACACCTTCACCGGTTACTAdomain)TATGCACTGGGTGCGGCAGGCCCCGGGCCAGGGGTTGGAATGGATGGGATGGATCAATCCAAACTCGGGTGGGACTAACT >VA58-ACGCCCAGAAGTTCCAAGGACGGGTGACCATGACTAGGGACACCTCGATCTCCACCGCATACATGGAGCTTAGCAGACTC21LCCGCTCCGACGATACCGCAGTCTACTATTGCGCGCGGGGAGAGTGGGACGGATCGTACTACTACGATTACTGGGGCCAGGG(M3)AACTCTGGTGACTGTTTCCTCGGGTGGAGGAGGTTCAGGCGGAGGCGGCTCGGGCGGGGGAGGATCTGGAGGAGGAGGGTCCGACATTGTGCTGACCCAAACTCCTTCGTCCCTGTCGGCCAGCGTGGGCGACCGCGTGACGATTACGTGCAGAGCTAGCCAATCCATCAATACTTACCTCAACTGGTACCAGCATAAGCCGGGGAAAGCACCAAAGCTGCTGATCTACGCCGCCTCATCCTTGCAGAGCGGTGTGCCTTCACGCTTTAGCGGATCGGGATCGGGAACGGATTTCACCCTGACTATCAGCTCCCTCCAGCCGGAGGATTTTGCGACCTACTACTGTCAGCAGAGCTTCTCACCGCTGACTTTCGGCGGCGGGACCAAGCTGGAAATCAAG113 M3ATGGCCCTCCCTGTCACCGCCCTGCTGCTTCCGCTGGCTCTTCTGCTCCACGCCGCTCGGCCCCAAGTCCAACTCGTCCA(Full) A >VA58-TCAGGAGCGGAAGTCAAAAAGCCCGGAGCTCCAGTGAAAGTGTCATGCAAGGCCTCCGGCTACACCTTCACCGGTTACTA21LCTATGCACTGGGTGCGGCAGGCCCCGGGCCAGGGGTTGGAATGGATGGGATGGATCAATCCAAACTCGGGTGGGACTAACT(M3)ACGCCCAGAAGTTCCAAGGACGGGTGACCATGACTAGGGACACCTCGATCTCCACCGCATACATGGAGCTTAGCAGACTCCGCTCCGACGATACCGCAGTCTACTATTGCGCGCGGGGAGAGTGGGACGGATCGTACTACTACGATTACTGGGGCCAGGGAACTCTGGTGACTGTTTCCTCGGGTGGAGGAGGTTCAGGCGGAGGCGGCTCGGGCGGGGGAGGATCTGGAGGAGGAGGGTCCGACATTGTGCTGACCCAAACTCCTTCGTCCCTGTCGGCCAGCGTGGGCGACCGCGTGACGATTACGTGCAGAGCTAGCCAATCCATCAATACTTACCTCAACTGGTACCAGCATAAGCCGGGGAAAGCACCAAAGCTGCTGATCTACGCCGCCTCATCCTTGCAGAGCGGTGTGCCTTCACGCTTTAGCGGATCGGGATCGGGAACGGATTTCACCCTGACTATCAGCTCCCTCCAGCCGGAGGATTTTGCGACCTACTACTGTCAGCAGAGCTTCTCACCGCTGACTTTCGGCGGCGGGACCAAGCTGGAAATCAAGACCACTACCCCAGCACCGAGGCCACCCACCCCGGCTCCTACCATCGCCTCCCAGCCTCTGTCCCTGCGTCCGGAGGCATGTAGACCCGCAGCTGGTGGGGCCGTGCATACCCGGGGTCTTGACTTCGCCTGCGATATCTACATTTGGGCCCCTCTGGCTGGTACTTGCGGGGTCCTGCTGCTTTCACTCGTGATCACTCTTTACTGTAAGCGCGGTCGGAAGAAGCTGCTGTACATCTTTAAGCAACCCTTCATGAGGCCTGTGCAGACTACTCAAGAGGAGGACGGCTGTTCATGCCGGTTCCCAGAGGAGGAGGAAGGCGGCTGCGAACTGCGCGTGAAATTCAGCCGCAGCGCAGATGCTCCAGCCTACAAGCAGGGGCAGAACCAGCTCTACAACGAACTCAATCTTGGTCGGAGAGAGGAGTACGACGTGCTGGACAAGCGGAGAGGACGGGACCCAGAAATGGGCGGGAAGCCGCGCAGAAAGAATCCCCAAGAGGGCCTGTACAACGAGCTCCAAAAGGATAAGATGGCAGAAGCCTATAGCGAGATTGGTATGAAAGGGGAACGCAGAAGAGGCAAAGGCCACGACGGACTGTACCAGGGACTCAGCACCGCCACCAAGGACACCTATGACGCTCTTCACATGCAGGCCCTGCCGCCTCGG 90 M4 CAAGTGCAACTCGTTGAA (ScFvTCAGGTGGAGGTTTGGTGCAACCCGGAGGATCTCTCAGACTGTCGTGTGCGGCGTCCGGGTTCACCTTTTCGTCCTACTGdomain)GATGCACTGGGTGCGCCAGGTGCCGGGAAAAGGACTGGTGTGGGTGTCCAGAATCAACACCGACGGGTCAACGACTACCT >DP37-ACGCAGATAGCGTGGAAGGTCGGTTCACCATTTCGCGGGACAACGCTAAAAACACTCTGTACCTTCAGATGAATTCACTG07ICCGCGATGACGACACCGCAGTCTACTACTGCGTCGGTGGACACTGGGCGGTCTGGGGACAGGGAACTACGGTGACTGTGTC(M4)CAGCGGCGGGGGAGGAAGCGGCGGAGGGGGGAGCGGAGGCGGAGGATCAGGAGGAGGCGGCTCCGATATCCAGATGACCCAGTCGCCATCGACCCTCTCCGCTAGCGTGGGGGATAGGGTCACTATCACTTGCCGAGCCAGCCAATCCATTAGCGACCGGCTTGCCTGGTACCAACAGAAACCTGGAAAGGCCCCGAAGCTGCTCATCTACAAGGCCTCGTCACTGGAGTCGGGAGTCCCGTCCCGCTTTTCCGGCTCGGGCTCAGGCACCGAGTTCACTCTGACCATCTCGAGCCTGCAGCCGGACGATTTCGCCGTGTATTACTGCCAGCAATACGGACATCTCCCAATGTACACGTTCGGTCAGGGCACCAAGGTCGAAATCAAG 114M4ATGGCCCTCCCTGTCACCGCCCTGCTGCTTCCGCTGGCTCTTCTGCTCCACGCCGCTCGGCCCCAAGTGCAACTCGTTGA >DP37-A 07ICTCAGGTGGAGGTTTGGTGCAACCCGGAGGATCTCTCAGACTGTCGTGTGCGGCGTCCGGGTTCACCTTTTCGTCCTACTG(M4)GATGCACTGGGTGCGCCAGGTGCCGGGAAAAGGACTGGTGTGGGTGTCCAGAATCAACACCGACGGGTCAACGACTACCTACGCAGATAGCGTGGAAGGTCGGTTCACCATTTCGCGGGACAACGCTAAAAACACTCTGTACCTTCAGATGAATTCACTGCGCGATGACGACACCGCAGTCTACTACTGCGTCGGTGGACACTGGGCGGTCTGGGGACAGGGAACTACGGTGACTGTGTCCAGCGGCGGGGGAGGAAGCGGCGGAGGGGGGAGCGGAGGCGGAGGATCAGGAGGAGGCGGCTCCGATATCCAGATGACCCAGTCGCCATCGACCCTCTCCGCTAGCGTGGGGGATAGGGTCACTATCACTTGCCGAGCCAGCCAATCCATTAGCGACCGGCTTGCCTGGTACCAACAGAAACCTGGAAAGGCCCCGAAGCTGCTCATCTACAAGGCCTCGTCACTGGAGTCGGGAGTCCCGTCCCGCTTTTCCGGCTCGGGCTCAGGCACCGAGTTCACTCTGACCATCTCGAGCCTGCAGCCGGACGATTTCGCCGTGTATTACTGCCAGCAATACGGACATCTCCCAATGTACACGTTCGGTCAGGGCACCAAGGTCGAAATCAAGACCACTACCCCAGCACCGAGGCCACCCACCCCGGCTCCTACCATCGCCTCCCAGCCTCTGTCCCTGCGTCCGGAGGCATGTAGACCCGCAGCTGGTGGGGCCGTGCATACCCGGGGTCTTGACTTCGCCTGCGATATCTACATTTGGGCCCCTCTGGCTGGTACTTGCGGGGTCCTGCTGCTTTCACTCGTGATCACTCTTTACTGTAAGCGCGGTCGGAAGAAGCTGCTGTACATCTTTAAGCAACCCTTCATGAGGCCTGTGCAGACTACTCAAGAGGAGGACGGCTGTTCATGCCGGTTCCCAGAGGAGGAGGAAGGCGGCTGCGAACTGCGCGTGAAATTCAGCCGCAGCGCAGATGCTCCAGCCTACAAGCAGGGGCAGAACCAGCTCTACAACGAACTCAATCTTGGTCGGAGAGAGGAGTACGACGTGCTGGACAAGCGGAGAGGACGGGACCCAGAAATGGGCGGGAAGCCGCGCAGAAAGAATCCCCAAGAGGGCCTGTACAACGAGCTCCAAAAGGATAAGATGGCAGAAGCCTATAGCGAGATTGGTATGAAAGGGGAACGCAGAAGAGGCAAAGGCCACGACGGACTGTACCAGGGACTCAGCACCGCCACCAAGGACACCTATGACGCTCTTCACATGCAGGCCCTGCCGCCTCGG 91 M5CAAGTCCAACTCGTTCAATCAGGCGCAGAAGTCGAAAAGCCCGGAGCATCAGTCAAAGTCTCTTGCAAGGCTTCCGGCTA(ScFv CACCTTCACGGACTACTAC domain)ATGCACTGGGTGCGCCAGGCTCCAGGCCAGGGACTGGAGTGGATGGGATGGATCAACCCGAATTCCGGGGGAACTAACTA >XP31-CGCCCAGAAGTTTCAGGGCCGGGTGACTATGACTCGCGATACCTCGATCTCGACTGCGTACATGGAGCTCAGCCGCCTCC20LCGGTCGGACGATACCGCCGTGTACTATTGTGCGTCGGGATGGGACTTCGACTACTGGGGGCAGGGCACTCTGGTCACTGTG(M5)TCAAGCGGAGGAGGTGGATCAGGTGGAGGTGGAAGCGGGGGAGGAGGTTCCGGCGGCGGAGGATCAGATATCGTGATGACGCAATCGCCTTCCTCGTTGTCCGCATCCGTGGGAGACAGGGTGACCATTACTTGCAGAGCGTCCCAGTCCATTCGGTACTACCTGTCGTGGTACCAGCAGAAGCCGGGGAAAGCCCCAAAACTGCTTATCTATACTGCCTCGATCCTCCAAAACGGCGTGCCATCAAGATTCAGCGGTTCGGGCAGCGGGACCGACTTTACCCTGACTATCAGCAGCCTGCAGCCGGAAGATTTCGCCACGTACTACTGCCTGCAAACCTACACCACCCCGGACTTCGGACCTGGAACCAAGGTGGAGATCAAG 115 M5ATGGCCCTCCCTGTCACCGCCCTGCTGCTTCCGCTGGCTCTTCTGCTCCACGCCGCTCGGCCCCAAGTCCAACTCGTTCA(Full)ATCAGGCGCAGAAGTCGAAAAGCCCGGAGCATCAGTCAAAGTCTCTTGCAAGGCTTCCGGCTACACCTTCACGGACTACT >XP31-AC 20LCATGCACTGGGTGCGCCAGGCTCCAGGCCAGGGACTGGAGTGGATGGGATGGATCAACCCGAATTCCGGGGGAACTAACTA(M5)CGCCCAGAAGTTTCAGGGCCGGGTGACTATGACTCGCGATACCTCGATCTCGACTGCGTACATGGAGCTCAGCCGCCTCCGGTCGGACGATACCGCCGTGTACTATTGTGCGTCGGGATGGGACTTCGACTACTGGGGGCAGGGCACTCTGGTCACTGTGTCAAGCGGAGGAGGTGGATCAGGTGGAGGTGGAAGCGGGGGAGGAGGTTCCGGCGGCGGAGGATCAGATATCGTGATGACGCAATCGCCTTCCTCGTTGTCCGCATCCGTGGGAGACAGGGTGACCATTACTTGCAGAGCGTCCCAGTCCATTCGGTACTACCTGTCGTGGTACCAGCAGAAGCCGGGGAAAGCCCCAAAACTGCTTATCTATACTGCCTCGATCCTCCAAAACGGCGTGCCATCAAGATTCAGCGGTTCGGGCAGCGGGACCGACTTTACCCTGACTATCAGCAGCCTGCAGCCGGAAGATTTCGCCACGTACTACTGCCTGCAAACCTACACCACCCCGGACTTCGGACCTGGAACCAAGGTGGAGATCAAGACCACTACCCCAGCACCGAGGCCACCCACCCCGGCTCCTACCATCGCCTCCCAGCCTCTGTCCCTGCGTCCGGAGGCATGTAGACCCGCAGCTGGTGGGGCCGTGCATACCCGGGGTCTTGACTTCGCCTGCGATATCTACATTTGGGCCCCTCTGGCTGGTACTTGCGGGGTCCTGCTGCTTTCACTCGTGATCACTCTTTACTGTAAGCGCGGTCGGAAGAAGCTGCTGTACATCTTTAAGCAACCCTTCATGAGGCCTGTGCAGACTACTCAAGAGGAGGACGGCTGTTCATGCCGGTTCCCAGAGGAGGAGGAAGGCGGCTGCGAACTGCGCGTGAAATTCAGCCGCAGCGCAGATGCTCCAGCCTACAAGCAGGGGCAGAACCAGCTCTACAACGAACTCAATCTTGGTCGGAGAGAGGAGTACGACGTGCTGGACAAGCGGAGAGGACGGGACCCAGAAATGGGCGGGAAGCCGCGCAGAAAGAATCCCCAAGAGGGCCTGTACAACGAGCTCCAAAAGGATAAGATGGCAGAAGCCTATAGCGAGATTGGTATGAAAGGGGAACGCAGAAGAGGCAAAGGCCACGACGGACTGTACCAGGGACTCAGCACCGCCACCAAGGACACCTATGACGCTCTTCACATGCAGGCCCTGCCGCCTCGG 92 M6CAAGTGCAACTCGTCCAGTCAGGTGCAGAAGTGAAGAAACCCGGAGCGTCAGTCAAAGTGTCATGCAAGGCGTCAGGCTA(ScFv CACCTTCACCAGCTACTAC domain)ATGCACTGGGTGCGGCAGGCCCCAGGCCAAGGCTTGGAGTGGATGGGAATCATTAACCCGTCAGGAGGCTCCACCTCCTA >FE10-CGCCCAGAAGTTTCAGGGAAGAGTGACGATGACTCGGGATACGTCGACCTCGACCGTGTACATGGAACTGAGCTCGCTGC06IDGCTCCGAGGACACTGCTGTGTACTACTGCGCACGGTACAGACTCATTGCCGTGGCAGGAGACTACTACTACTATGGCATG(M6)GACGTCTGGGGGCAGGGCACTATGGTCACTGTGTCGTCCGGCGGAGGAGGCTCGGGTGGAGGAGGTAGCGGAGGAGGGGGAAGCGGAGGGGGGGGCTCCGATATCCAGATGACTCAGTCGCCTTCCTCCGTGTCGGCCTCGGTTGGAGATCGCGTCACCATCACTTGTCGAGCTTCCCAAGGAGTCGGTAGGTGGCTGGCGTGGTACCAGCAAAAGCCGGGAACTGCCCCGAAGCTCCTGATCTACGCGGCTAGCACCCTGCAGTCGGGAGTGCCATCCCGCTTCAGCGGATCTGGGTCAGGTACCGACTTCACCCTTACGATCAACAATCTCCAGCCGGAGGACTTTGCCACCTATTACTGCCAACAGGCCAACAGCTTCCCTCTGACTTTCGGAGGGGGCACTCGCCTGGAAATCAAG 116 M6ATGGCCCTCCCTGTCACCGCCCTGCTGCTTCCGCTGGCTCTTCTGCTCCACGCCGCTCGGCCCCAAGTGCAACTCGTCCA(Full)GTCAGGTGCAGAAGTGAAGAAACCCGGAGCGTCAGTCAAAGTGTCATGCAAGGCGTCAGGCTACACCTTCACCAGCTACT >FE10-AC 06IDATGCACTGGGTGCGGCAGGCCCCAGGCCAAGGCTTGGAGTGGATGGGAATCATTAACCCGTCAGGAGGCTCCACCTCCTA(M6)CGCCCAGAAGTTTCAGGGAAGAGTGACGATGACTCGGGATACGTCGACCTCGACCGTGTACATGGAACTGAGCTCGCTGCGCTCCGAGGACACTGCTGTGTACTACTGCGCACGGTACAGACTCATTGCCGTGGCAGGAGACTACTACTACTATGGCATGGACGTCTGGGGGCAGGGCACTATGGTCACTGTGTCGTCCGGCGGAGGAGGCTCGGGTGGAGGAGGTAGCGGAGGAGGGGGAAGCGGAGGGGGGGGCTCCGATATCCAGATGACTCAGTCGCCTTCCTCCGTGTCGGCCTCGGTTGGAGATCGCGTCACCATCACTTGTCGAGCTTCCCAAGGAGTCGGTAGGTGGCTGGCGTGGTACCAGCAAAAGCCGGGAACTGCCCCGAAGCTCCTGATCTACGCGGCTAGCACCCTGCAGTCGGGAGTGCCATCCCGCTTCAGCGGATCTGGGTCAGGTACCGACTTCACCCTTACGATCAACAATCTCCAGCCGGAGGACTTTGCCACCTATTACTGCCAACAGGCCAACAGCTTCCCTCTGACTTTCGGAGGGGGCACTCGCCTGGAAATCAAGACCACTACCCCAGCACCGAGGCCACCCACCCCGGCTCCTACCATCGCCTCCCAGCCTCTGTCCCTGCGTCCGGAGGCATGTAGACCCGCAGCTGGTGGGGCCGTGCATACCCGGGGTCTTGACTTCGCCTGCGATATCTACATTTGGGCCCCTCTGGCTGGTACTTGCGGGGTCCTGCTGCTTTCACTCGTGATCACTCTTTACTGTAAGCGCGGTCGGAAGAAGCTGCTGTACATCTTTAAGCAACCCTTCATGAGGCCTGTGCAGACTACTCAAGAGGAGGACGGCTGTTCATGCCGGTTCCCAGAGGAGGAGGAAGGCGGCTGCGAACTGCGCGTGAAATTCAGCCGCAGCGCAGATGCTCCAGCCTACAAGCAGGGGCAGAACCAGCTCTACAACGAACTCAATCTTGGTCGGAGAGAGGAGTACGACGTGCTGGACAAGCGGAGAGGACGGGACCCAGAAATGGGCGGGAAGCCGCGCAGAAAGAATCCCCAAGAGGGCCTGTACAACGAGCTCCAAAAGGATAAGATGGCAGAAGCCTATAGCGAGATTGGTATGAAAGGGGAACGCAGAAGAGGCAAAGGCCACGACGGACTGTACCAGGGACTCAGCACCGCCACCAAGGACACCTATGACGCTCTTCACATGCAGGCCCTGCCGCCTCGG 93 M7CAAGTGCAATTGGTTCAA (ScFvTCAGGAGGAGGAGTGGTGCAACCTGGAAGATCTCTCAGACTGTCGTGTGCGGCATCGGGATTCACTTTCTCATCATACGCdomain)AATGCACTGGGTCCGCCAGGCCCCGGGCAAAGGCTTGGAATGGGTGGCGGTCATTTCATACGACGGCTCGAACAAGTACT >VE12-ACGCTGACAGCGTGAAGGGACGCTTTACTATTTCCCGGGACAATTCGAAGAACACTCTGTACCTCCAGATGAACTCCCTT01CDAGGGCTGAGGACACCGCCGTCTACTACTGCGCACGCTGGAAAGTGTCGTCCAGCTCCCCAGCTTTTGACTACTGGGGACA(M7)GGGAACCCTTGTGACCGTGTCGTCCGGTGGAGGGGGAAGCGGCGGAGGGGGATCAGGTGGCGGCGGATCGGGAGGCGGGGGATCAGAAATCGTGCTGACTCAGTCCCCGGCCACGCTGTCTCTCAGCCCGGGAGAGAGAGCGATCCTGTCCTGCCGCGCCTCGCAGAGCGTGTACACTAAGTACCTGGGGTGGTACCAGCAGAAACCGGGTCAAGCGCCTCGGCTGCTGATCTACGATGCCTCCACCCGGGCCACCGGAATCCCCGATCGGTTCTCCGGCAGCGGCTCGGGAACTGATTTCACGCTGACCATCAATCGCCTGGAGCCGGAAGATTTCGCCGTCTATTACTGCCAGCATTACGGCGGGAGCCCACTCATCACCTTCGGTCAAGGAACCCGACTCGAAATCAAG 117 M7ATGGCCCTCCCTGTCACCGCCCTGCTGCTTCCGCTGGCTCTTCTGCTCCACGCCGCTCGGCCCCAAGTGCAATTGGTTCA(Full) A >VE12-TCAGGAGGAGGAGTGGTGCAACCTGGAAGATCTCTCAGACTGTCGTGTGCGGCATCGGGATTCACTTTCTCATCATACGC01CDAATGCACTGGGTCCGCCAGGCCCCGGGCAAAGGCTTGGAATGGGTGGCGGTCATTTCATACGACGGCTCGAACAAGTACT(M7)ACGCTGACAGCGTGAAGGGACGCTTTACTATTTCCCGGGACAATTCGAAGAACACTCTGTACCTCCAGATGAACTCCCTTAGGGCTGAGGACACCGCCGTCTACTACTGCGCACGCTGGAAAGTGTCGTCCAGCTCCCCAGCTTTTGACTACTGGGGACAGGGAACCCTTGTGACCGTGTCGTCCGGTGGAGGGGGAAGCGGCGGAGGGGGATCAGGTGGCGGCGGATCGGGAGGCGGGGGATCAGAAATCGTGCTGACTCAGTCCCCGGCCACGCTGTCTCTCAGCCCGGGAGAGAGAGCGATCCTGTCCTGCCGCGCCTCGCAGAGCGTGTACACTAAGTACCTGGGGTGGTACCAGCAGAAACCGGGTCAAGCGCCTCGGCTGCTGATCTACGATGCCTCCACCCGGGCCACCGGAATCCCCGATCGGTTCTCCGGCAGCGGCTCGGGAACTGATTTCACGCTGACCATCAATCGCCTGGAGCCGGAAGATTTCGCCGTCTATTACTGCCAGCATTACGGCGGGAGCCCACTCATCACCTTCGGTCAAGGAACCCGACTCGAAATCAAGACCACTACCCCAGCACCGAGGCCACCCACCCCGGCTCCTACCATCGCCTCCCAGCCTCTGTCCCTGCGTCCGGAGGCATGTAGACCCGCAGCTGGTGGGGCCGTGCATACCCGGGGTCTTGACTTCGCCTGCGATATCTACATTTGGGCCCCTCTGGCTGGTACTTGCGGGGTCCTGCTGCTTTCACTCGTGATCACTCTTTACTGTAAGCGCGGTCGGAAGAAGCTGCTGTACATCTTTAAGCAACCCTTCATGAGGCCTGTGCAGACTACTCAAGAGGAGGACGGCTGTTCATGCCGGTTCCCAGAGGAGGAGGAAGGCGGCTGCGAACTGCGCGTGAAATTCAGCCGCAGCGCAGATGCTCCAGCCTACAAGCAGGGGCAGAACCAGCTCTACAACGAACTCAATCTTGGTCGGAGAGAGGAGTACGACGTGCTGGACAAGCGGAGAGGACGGGACCCAGAAATGGGCGGGAAGCCGCGCAGAAAGAATCCCCAAGAGGGCCTGTACAACGAGCTCCAAAAGGATAAGATGGCAGAAGCCTATAGCGAGATTGGTATGAAAGGGGAACGCAGAAGAGGCAAAGGCCACGACGGACTGTACCAGGGACTCAGCACCGCCACCAAGGACACCTATGACGCTCTTCACATGCAGGCCCTGCCGCCTCGG 94 M8 CAAGTCCAACTCCAGCAG (ScFvTCAGGTGCAGAAGTCAAAAAGCCAGGAGCATCCGTGAAGGTTTCGTGCAAGACTTCCGGCTACCCTTTTACCGGGTACTCdomain)cCTCCATTGGGTGAGACAAGCACCGGGCCAGGGACTGGAGTGGATGGGATGGATCAACCCAAATTCGGGCGGCACCAACT >LE13-ATGCGCAGAAGTTCCAGGGACGGGTGACCATGACTCGCGACACTTCGATCTCCACTGCCTACATGGAGCTGTCCCGCTTG05XDAGATCTGACGACACGGCCGTCTACTACTGCGCCCGGGATCACTACGGAGGTAATTCGCTGTTCTACTGGGGGCAGGGAAC(M8)CCTTGTGACTGTGTCCTCGGGTGGTGGAGGGTCAGGAGGCGGAGGCTCAGGGGGAGGAGGTAGCGGAGGAGGCGGATCAGACATCCAACTGACCCAGTCACCATCCTCCATCTCGGCTAGCGTCGGAGACACCGTGTCGATTACTTGTAGGGCCTCCCAAGACTCAGGGACGTGGCTGGCGTGGTATCAGCAAAAACCGGGCAAAGCTCCGAACCTGTTGATGTACGACGCCAGCACCCTCGAAGATGGAGTGCCTAGCCGCTTCAGCGGAAGCGCCTCGGGCACTGAATTCACGCTGACTGTGAATCGGCTCCAGCCGGAGGATTCGGCGACCTACTACTGCCAGCAGTACAACAGCTACCCCCTGACCTTTGGAGGCGGGACCAAGGTGGATATCAAG118 M8ATGGCCCTCCCTGTCACCGCCCTGCTGCTTCCGCTGGCTCTTCTGCTCCACGCCGCTCGGCCCCAAGTCCAACTCCAGCA(Full) G >LE13-TCAGGTGCAGAAGTCAAAAAGCCAGGAGCATCCGTGAAGGTTTCGTGCAAGACTTCCGGCTACCCTTTTACCGGGTACTC05XDCCTCCATTGGGTGAGACAAGCACCGGGCCAGGGACTGGAGTGGATGGGATGGATCAACCCAAATTCGGGCGGCACCAACT(M8)ATGCGCAGAAGTTCCAGGGACGGGTGACCATGACTCGCGACACTTCGATCTCCACTGCCTACATGGAGCTGTCCCGCTTGAGATCTGACGACACGGCCGTCTACTACTGCGCCCGGGATCACTACGGAGGTAATTCGCTGTTCTACTGGGGGCAGGGAACCCTTGTGACTGTGTCCTCGGGTGGTGGAGGGTCAGGAGGCGGAGGCTCAGGGGGAGGAGGTAGCGGAGGAGGCGGATCAGACATCCAACTGACCCAGTCACCATCCTCCATCTCGGCTAGCGTCGGAGACACCGTGTCGATTACTTGTAGGGCCTCCCAAGACTCAGGGACGTGGCTGGCGTGGTATCAGCAAAAACCGGGCAAAGCTCCGAACCTGTTGATGTACGACGCCAGCACCCTCGAAGATGGAGTGCCTAGCCGCTTCAGCGGAAGCGCCTCGGGCACTGAATTCACGCTGACTGTGAATCGGCTCCAGCCGGAGGATTCGGCGACCTACTACTGCCAGCAGTACAACAGCTACCCCCTGACCTTTGGAGGCGGGACCAAGGTGGATATCAAGACCACTACCCCAGCACCGAGGCCACCCACCCCGGCTCCTACCATCGCCTCCCAGCCTCTGTCCCTGCGTCCGGAGGCATGTAGACCCGCAGCTGGTGGGGCCGTGCATACCCGGGGTCTTGACTTCGCCTGCGATATCTACATTTGGGCCCCTCTGGCTGGTACTTGCGGGGTCCTGCTGCTTTCACTCGTGATCACTCTTTACTGTAAGCGCGGTCGGAAGAAGCTGCTGTACATCTTTAAGCAACCCTTCATGAGGCCTGTGCAGACTACTCAAGAGGAGGACGGCTGTTCATGCCGGTTCCCAGAGGAGGAGGAAGGCGGCTGCGAACTGCGCGTGAAATTCAGCCGCAGCGCAGATGCTCCAGCCTACAAGCAGGGGCAGAACCAGCTCTACAACGAACTCAATCTTGGTCGGAGAGAGGAGTACGACGTGCTGGACAAGCGGAGAGGACGGGACCCAGAAATGGGCGGGAAGCCGCGCAGAAAGAATCCCCAAGAGGGCCTGTACAACGAGCTCCAAAAGGATAAGATGGCAGAAGCCTATAGCGAGATTGGTATGAAAGGGGAACGCAGAAGAGGCAAAGGCCACGACGGACTGTACCAGGGACTCAGCACCGCCACCAAGGACACCTATGACGCTCTTCACATGCAGGCCCTGCCGCCTCGG 95 M9 CAAGTGCAACTCGTCCAG (ScFvTCAGGTGCAGAAGTGAAGAAACCAGGAGCGTCCGTCGAAGTGTCGTGTAAGGCGTCCGGCTACACTTTCACCTCGTACTAdomain)CATGCACTGGGTGCGGCAGGCCCCGGGACAAGGCCTCGAATGGATGGGAATCATCAACCCGAGCGGAGGCTCGACTGGTT >BE15-ACGCCCAGAAGTTCCAGGGAAGGGTGACGATGACCCGCGATACCTCGACTTCGACCGTTCATATGGAGCTCTCGTCCCTG00SDCGGAGCGAGGACACTGCTGTCTACTATTGCGCGCGGGGAGGATACTCTAGCTCCTCCGATGCATTTGACATTTGGGGCCA(M9)GGGAACTATGGTGACCGTGTCATCAGGCGGAGGTGGATCAGGAGGAGGAGGGTCGGGAGGGGGAGGCAGCGGCGGGGGTGGGTCGGACATTCAGATGACGCAGTCCCCTCCTAGCCTGAGCGCCTCGGTGGGTGACAGAGTGACCATCACTTGCAGAGCCTCGCAAGACATCTCCTCCGCATTGGCTTGGTACCAGCAAAAGCCGGGCACTCCGCCGAAACTGCTCATCTACGATGCCTCCTCACTGGAGTCAGGAGTCCCATCTCGCTTCTCGGGGTCAGGAAGCGGCACCGATTTTACCCTTACCATCTCCAGCCTGCAGCCCGAGGACTTCGCCACGTACTACTGCCAACAGTTCAGCTCCTACCCACTGACCTTCGGGGGCGGAACTCGCCTGGAAATCAAG 119 M9ATGGCCCTCCCTGTCACCGCCCTGCTGCTTCCGCTGGCTCTTCTGCTCCACGCCGCTCGGCCCCAAGTGCAACTCGTCCA(Full) G >BE15-TCAGGTGCAGAAGTGAAGAAACCAGGAGCGTCCGTCGAAGTGTCGTGTAAGGCGTCCGGCTACACTTTCACCTCGTACTA00SDCATGCACTGGGTGCGGCAGGCCCCGGGACAAGGCCTCGAATGGATGGGAATCATCAACCCGAGCGGAGGCTCGACTGGTT(M9)ACGCCCAGAAGTTCCAGGGAAGGGTGACGATGACCCGCGATACCTCGACTTCGACCGTTCATATGGAGCTCTCGTCCCTGCGGAGCGAGGACACTGCTGTCTACTATTGCGCGCGGGGAGGATACTCTAGCTCCTCCGATGCATTTGACATTTGGGGCCAGGGAACTATGGTGACCGTGTCATCAGGCGGAGGTGGATCAGGAGGAGGAGGGTCGGGAGGGGGAGGCAGCGGCGGGGGTGGGTCGGACATTCAGATGACGCAGTCCCCTCCTAGCCTGAGCGCCTCGGTGGGTGACAGAGTGACCATCACTTGCAGAGCCTCGCAAGACATCTCCTCCGCATTGGCTTGGTACCAGCAAAAGCCGGGCACTCCGCCGAAACTGCTCATCTACGATGCCTCCTCACTGGAGTCAGGAGTCCCATCTCGCTTCTCGGGGTCAGGAAGCGGCACCGATTTTACCCTTACCATCTCCAGCCTGCAGCCCGAGGACTTCGCCACGTACTACTGCCAACAGTTCAGCTCCTACCCACTGACCTTCGGGGGCGGAACTCGCCTGGAAATCAAGACCACTACCCCAGCACCGAGGCCACCCACCCCGGCTCCTACCATCGCCTCCCAGCCTCTGTCCCTGCGTCCGGAGGCATGTAGACCCGCAGCTGGTGGGGCCGTGCATACCCGGGGTCTTGACTTCGCCTGCGATATCTACATTTGGGCCCCTCTGGCTGGTACTTGCGGGGTCCTGCTGCTTTCACTCGTGATCACTCTTTACTGTAAGCGCGGTCGGAAGAAGCTGCTGTACATCTTTAAGCAACCCTTCATGAGGCCTGTGCAGACTACTCAAGAGGAGGACGGCTGTTCATGCCGGTTCCCAGAGGAGGAGGAAGGCGGCTGCGAACTGCGCGTGAAATTCAGCCGCAGCGCAGATGCTCCAGCCTACAAGCAGGGGCAGAACCAGCTCTACAACGAACTCAATCTTGGTCGGAGAGAGGAGTACGACGTGCTGGACAAGCGGAGAGGACGGGACCCAGAAATGGGCGGGAAGCCGCGCAGAAAGAATCCCCAAGAGGGCCTGTACAACGAGCTCCAAAAGGATAAGATGGCAGAAGCCTATAGCGAGATTGGTATGAAAGGGGAACGCAGAAGAGGCAAAGGCCACGACGGACTGTACCAGGGACTCAGCACCGCCACCAAGGACACCTATGACGCTCTTCACATGCAGGCCCTGCCGCCTCGG 96 M10CAAGTGCAACTCGTCCAGAGCGGAGCAGAAGTCAAGAAGCCAGGAGCGTCAGTGAAAGTGTCATGCAAGGCCAGCGGCTA(ScFv TACCTTTACTTCGTATGGG domain)ATCTCCTGGGTGCGGCAGGCACCGGGCCAAGGACTGGAGTGGATGGGATGGATCTCAGCCTACAACGGTAACACCAACTA >RE16-CGCCCAGAAGCTGCAAGGACGCGTGACCATGACTACTGATACGAGCACCTCCACTGCCTACATGGAATTGCGGTCCCTTC05MDGGTCGGACGATACTGCTGTGTACTACTGCGCAAGAGTCGCCGGAGGGATCTACTACTACTACGGCATGGACGTCTGGGGA(M10)CAGGGAACCACCATTACGGTGTCGAGCGGAGGGGGAGGCTCGGGGGGAGGAGGAAGCGGAGGTGGCGGCTCCGGGGGCGGCGGATCGGACATTGTGATGACCCAGACTCCTGACTCCCTGGCTGTTTCGTTGGGAGAGCGCGCGACTATCTCGTGTAAGTCCAGCCACTCAGTCCTGTACAATCGCAATAACAAGAACTACCTCGCGTGGTACCAGCAAAAACCGGGTCAGCCGCCTAAACTCCTGTTCTACTGGGCCTCCACCAGAAAGAGCGGGGTGCCAGATCGATTCTCTGGATCAGGATCAGGTACCGACTTTACGCTGACCATCTCGTCCCTGCAGCCGGAGGATTTCGCGACTTACTTCTGCCAGCAGACTCAGACTTTCCCCCTCACCTTCGGTCAAGGCACCAGGCTGGAAATCAAT 120 M10ATGGCCCTCCCTGTCACCGCCCTGCTGCTTCCGCTGGCTCTTCTGCTCCACGCCGCTCGGCCCCAAGTGCAACTCGTCCA(Full)GAGCGGAGCAGAAGTCAAGAAGCCAGGAGCGTCAGTGAAAGTGTCATGCAAGGCCAGCGGCTATACCTTTACTTCGTATG >RE16-GG 05MDATCTCCTGGGTGCGGCAGGCACCGGGCCAAGGACTGGAGTGGATGGGATGGATCTCAGCCTACAACGGTAACACCAACTA(M10)CGCCCAGAAGCTGCAAGGACGCGTGACCATGACTACTGATACGAGCACCTCCACTGCCTACATGGAATTGCGGTCCCTTCGGTCGGACGATACTGCTGTGTACTACTGCGCAAGAGTCGCCGGAGGGATCTACTACTACTACGGCATGGACGTCTGGGGACAGGGAACCACCATTACGGTGTCGAGCGGAGGGGGAGGCTCGGGGGGAGGAGGAAGCGGAGGTGGCGGCTCCGGGGGCGGCGGATCGGACATTGTGATGACCCAGACTCCTGACTCCCTGGCTGTTTCGTTGGGAGAGCGCGCGACTATCTCGTGTAAGTCCAGCCACTCAGTCCTGTACAATCGCAATAACAAGAACTACCTCGCGTGGTACCAGCAAAAACCGGGTCAGCCGCCTAAACTCCTGTTCTACTGGGCCTCCACCAGAAAGAGCGGGGTGCCAGATCGATTCTCTGGATCAGGATCAGGTACCGACTTTACGCTGACCATCTCGTCCCTGCAGCCGGAGGATTTCGCGACTTACTTCTGCCAGCAGACTCAGACTTTCCCCCTCACCTTCGGTCAAGGCACCAGGCTGGAAATCAATACCACTACCCCAGCACCGAGGCCACCCACCCCGGCTCCTACCATCGCCTCCCAGCCTCTGTCCCTGCGTCCGGAGGCATGTAGACCCGCAGCTGGTGGGGCCGTGCATACCCGGGGTCTTGACTTCGCCTGCGATATCTACATTTGGGCCCCTCTGGCTGGTACTTGCGGGGTCCTGCTGCTTTCACTCGTGATCACTCTTTACTGTAAGCGCGGTCGGAAGAAGCTGCTGTACATCTTTAAGCAACCCTTCATGAGGCCTGTGCAGACTACTCAAGAGGAGGACGGCTGTTCATGCCGGTTCCCAGAGGAGGAGGAAGGCGGCTGCGAACTGCGCGTGAAATTCAGCCGCAGCGCAGATGCTCCAGCCTACAAGCAGGGGCAGAACCAGCTCTACAACGAACTCAATCTTGGTCGGAGAGAGGAGTACGACGTGCTGGACAAGCGGAGAGGACGGGACCCAGAAATGGGCGGGAAGCCGCGCAGAAAGAATCCCCAAGAGGGCCTGTACAACGAGCTCCAAAAGGATAAGATGGCAGAAGCCTATAGCGAGATTGGTATGAAAGGGGAACGCAGAAGAGGCAAAGGCCACGACGGACTGTACCAGGGACTCAGCACCGCCACCAAGGACACCTATGACGCTCTTCACATGCAGGCCCTGCCGCCTCGG 97 M11CAAGTCCAATTGCAGCAGAGCGGAGCAGAAGTGAAGAAGCCAGGAGCGTCAGTCAAAGTGTCGTGTAAGGCGTCAGGATA(ScFv CACCTTCACGGGATACTAC domain)ATGCACTGGGTGCGCCAGGCCCCGGGCCAAGGACTCGAGTGGATGGGCTGGATCAACCCTAACTCTGGAGGCACCAACTA >NE10-CGCCCAGAATTTCCAAGGCAGAGTGACCATGACCCGGGACACCTCCATCTCGACTGCCTATATGGAACTGCGGCGGCTGC19WDGCTCGGACGATACTGCTGTGTATTACTGCGCCAGCGGCTGGGACTTTGACTACTGGGGACAGGGTACTCTGGTGACTGTT(M11)TCCTCGGGAGGAGGCGGATCGGGTGGAGGAGGTAGCGGGGGAGGGGGGTCGGGAGGCGGAGGCAGCGATATTCGCATGACTCAATCGCCGTCCTCCCTGAGCGCTAGCGTGGGAGATCGAGTCACCATCACTTGCAGAGCGTCACAGTCGATTCGCTACTACCTGTCCTGGTACCAGCAGAAACCGGGAAAGGCACCAAAGCTTCTGATCTACACGGCCTCCATCCTGCAAAATGGTGTCCCATCAAGGTTCTCCGGGTCAGGGAGCGGCACTGACTTCACTCTCACCATCTCCTCACTCCAGCCCGAGGACTTTGCAACCTACTACTGCCTCCAGACGTACACCACCCCGGATTTCGGTCCTGGAACCAAGGTGGAAATCAAA 121 M11ATGGCCCTCCCTGTCACCGCCCTGCTGCTTCCGCTGGCTCTTCTGCTCCACGCCGCTCGGCCCCAAGTCCAATTGCAGCA(Full)GAGCGGAGCAGAAGTGAAGAAGCCAGGAGCGTCAGTCAAAGTGTCGTGTAAGGCGTCAGGATACACCTTCACGGGATACT >NE10-AC 19WDATGCACTGGGTGCGCCAGGCCCCGGGCCAAGGACTCGAGTGGATGGGCTGGATCAACCCTAACTCTGGAGGCACCAACTA(M11)CGCCCAGAATTTCCAAGGCAGAGTGACCATGACCCGGGACACCTCCATCTCGACTGCCTATATGGAACTGCGGCGGCTGCGCTCGGACGATACTGCTGTGTATTACTGCGCCAGCGGCTGGGACTTTGACTACTGGGGACAGGGTACTCTGGTGACTGTTTCCTCGGGAGGAGGCGGATCGGGTGGAGGAGGTAGCGGGGGAGGGGGGTCGGGAGGCGGAGGCAGCGATATTCGCATGACTCAATCGCCGTCCTCCCTGAGCGCTAGCGTGGGAGATCGAGTCACCATCACTTGCAGAGCGTCACAGTCGATTCGCTACTACCTGTCCTGGTACCAGCAGAAACCGGGAAAGGCACCAAAGCTTCTGATCTACACGGCCTCCATCCTGCAAAATGGTGTCCCATCAAGGTTCTCCGGGTCAGGGAGCGGCACTGACTTCACTCTCACCATCTCCTCACTCCAGCCCGAGGACTTTGCAACCTACTACTGCCTCCAGACGTACACCACCCCGGATTTCGGTCCTGGAACCAAGGTGGAAATCAAAACCACTACCCCAGCACCGAGGCCACCCACCCCGGCTCCTACCATCGCCTCCCAGCCTCTGTCCCTGCGTCCGGAGGCATGTAGACCCGCAGCTGGTGGGGCCGTGCATACCCGGGGTCTTGACTTCGCCTGCGATATCTACATTTGGGCCCCTCTGGCTGGTACTTGCGGGGTCCTGCTGCTTTCACTCGTGATCACTCTTTACTGTAAGCGCGGTCGGAAGAAGCTGCTGTACATCTTTAAGCAACCCTTCATGAGGCCTGTGCAGACTACTCAAGAGGAGGACGGCTGTTCATGCCGGTTCCCAGAGGAGGAGGAAGGCGGCTGCGAACTGCGCGTGAAATTCAGCCGCAGCGCAGATGCTCCAGCCTACAAGCAGGGGCAGAACCAGCTCTACAACGAACTCAATCTTGGTCGGAGAGAGGAGTACGACGTGCTGGACAAGCGGAGAGGACGGGACCCAGAAATGGGCGGGAAGCCGCGCAGAAAGAATCCCCAAGAGGGCCTGTACAACGAGCTCCAAAAGGATAAGATGGCAGAAGCCTATAGCGAGATTGGTATGAAAGGGGAACGCAGAAGAGGCAAAGGCCACGACGGACTGTACCAGGGACTCAGCACCGCCACCAAGGACACCTATGACGCTCTTCACATGCAGGCCCTGCCGCCTCGG 98 M12 CAAGTCCAACTCGTCCAA (ScFvAGCGGAGCAGAAGTCAAAAAGCCAGGAGCGTCGGTGAAAGTGTCTTGCAAAGCCAGCGGCTACACCTTCACGGGTTACTAdomain)CATGCACTGGGTGCGCCAGGCGCCGGGCCAGGGGCTGGAGTGGATGGGCCGGATTAACCCTAACAGCGGGGGAACTAATT >DE12-ACGCTCAGAAGTTCCAGGGTAGAGTCACCATGACTACGGACACTTCCACTTCCACCGCCTATATGGAACTGCGCTCCCTC14RDCGCTCAGATGATACTGCCGTGTATTACTGCGCGCGGACTACCACGTCATACGCATTTGACATCTGGGGCCAGGGAACTAT(M12)GGTGACCGTGAGCTCGGGCGGAGGCGGTTCAGGGGGAGGAGGAAGCGGAGGAGGAGGATCGGGAGGAGGTGGCTCCGATATCCAGCTGACTCAGTCCCCGAGCACCCTGTCGGCGTCGGTGGGGGACAGGGTTACCATCACCTGTAGAGCTTCCCAATCCATTTCGACTTGGCTGGCCTGGTACCAGCAAAAGCCGGGAAAGGCCCCTAATTTGCTTATCTACAAGGCATCGACCCTCGAAAGCGGTGTGCCCTCCCGGTTTTCGGGATCAGGATCAGGGACCGAGTTCACCCTGACCATCTCATCCCTCCAGCCGGACGACTTCGCCACTTACTACTGCCAGCAGTACAACACCTACTCGCCATACACTTTCGGCCAAGGCACCAAGCTGGAGATCAAG122 M12ATGGCCCTCCCTGTCACCGCCCTGCTGCTTCCGCTGGCTCTTCTGCTCCACGCCGCTCGGCCCCAAGTCCAACTCGTCCA(Full) A >DE12-AGCGGAGCAGAAGTCAAAAAGCCAGGAGCGTCGGTGAAAGTGTCTTGCAAAGCCAGCGGCTACACCTTCACGGGTTACTA14RDCATGCACTGGGTGCGCCAGGCGCCGGGCCAGGGGCTGGAGTGGATGGGCCGGATTAACCCTAACAGCGGGGGAACTAATT(M12)ACGCTCAGAAGTTCCAGGGTAGAGTCACCATGACTACGGACACTTCCACTTCCACCGCCTATATGGAACTGCGCTCCCTCCGCTCAGATGATACTGCCGTGTATTACTGCGCGCGGACTACCACGTCATACGCATTTGACATCTGGGGCCAGGGAACTATGGTGACCGTGAGCTCGGGCGGAGGCGGTTCAGGGGGAGGAGGAAGCGGAGGAGGAGGATCGGGAGGAGGTGGCTCCGATATCCAGCTGACTCAGTCCCCGAGCACCCTGTCGGCGTCGGTGGGGGACAGGGTTACCATCACCTGTAGAGCTTCCCAATCCATTTCGACTTGGCTGGCCTGGTACCAGCAAAAGCCGGGAAAGGCCCCTAATTTGCTTATCTACAAGGCATCGACCCTCGAAAGCGGTGTGCCCTCCCGGTTTTCGGGATCAGGATCAGGGACCGAGTTCACCCTGACCATCTCATCCCTCCAGCCGGACGACTTCGCCACTTACTACTGCCAGCAGTACAACACCTACTCGCCATACACTTTCGGCCAAGGCACCAAGCTGGAGATCAAGACCACTACCCCAGCACCGAGGCCACCCACCCCGGCTCCTACCATCGCCTCCCAGCCTCTGTCCCTGCGTCCGGAGGCATGTAGACCCGCAGCTGGTGGGGCCGTGCATACCCGGGGTCTTGACTTCGCCTGCGATATCTACATTTGGGCCCCTCTGGCTGGTACTTGCGGGGTCCTGCTGCTTTCACTCGTGATCACTCTTTACTGTAAGCGCGGTCGGAAGAAGCTGCTGTACATCTTTAAGCAACCCTTCATGAGGCCTGTGCAGACTACTCAAGAGGAGGACGGCTGTTCATGCCGGTTCCCAGAGGAGGAGGAAGGCGGCTGCGAACTGCGCGTGAAATTCAGCCGCAGCGCAGATGCTCCAGCCTACAAGCAGGGGCAGAACCAGCTCTACAACGAACTCAATCTTGGTCGGAGAGAGGAGTACGACGTGCTGGACAAGCGGAGAGGACGGGACCCAGAAATGGGCGGGAAGCCGCGCAGAAAGAATCCCCAAGAGGGCCTGTACAACGAGCTCCAAAAGGATAAGATGGCAGAAGCCTATAGCGAGATTGGTATGAAAGGGGAACGCAGAAGAGGCAAAGGCCACGACGGACTGTACCAGGGACTCAGCACCGCCACCAAGGACACCTATGACGCTCTTCACATGCAGGCCCTGCCGCCTCGG 99 M13CAAGTTCAACTCGTGCAATCAGGTGGAGGACTCGTCAAACCCGGAGGATCATTGAGACTGTCATGCGAAGCGAGCGGTTT(ScFv TATCTTCTCCGATTACTAT domain)ATGGGATGGATTCGGCAGGCCCCGGGAAAGGGACTCGAATGGGTGTCATACATCGGAAGGTCAGGCTCGTCCATGTACTA >TE13-CGCAGACTCGGTGAAAGGCAGATTCACCTTTAGCCGGGACAACGCCAAGAATTCCCTCTACTTGCAGATGAACAGCCTGC19LDGAGCCGAGGATACTGCTGTCTACTACTGTGCCGCGTCGCCGGTGGTGGCAGCTACTGAAGATTTCCAGCACTGGGGACAG(M13)GGAACTCTGGTCACGGTGTCGAGCGGTGGGGGCGGAAGCGGAGGCGGAGGATCGGGCGGCGGAGGTTCGGGGGGGGGAGGGTCTGACATCGTGATGACCCAAACCCCAGCCACCCTGAGCCTCTCCCCTGGAGAGCGCGCGACTCTTTCGTGCCGCGCTTCCCAGTCAGTGACCAGCAATTACTTGGCTTGGTACCAACAGAAGCCGGGACAGGCGCCACGGCTGCTGCTTTTTGGTGCCAGCACTCGCGCCACCGGAATCCCGGATCGCTTCTCGGGCTCAGGGTCCGGGACGGACTTCACCCTGACTATCAACCGGCTGGAACCTGAGGACTTCGCGATGTACTACTGCCAGCAGTACGGCTCCGCACCAGTCACTTTCGGACAAGGCACCAAGCTGGAGATCAAG 123 M13ATGGCCCTCCCTGTCACCGCCCTGCTGCTTCCGCTGGCTCTTCTGCTCCACGCCGCTCGGCCCCAAGTTCAACTCGTGCA(Full)ATCAGGTGGAGGACTCGTCAAACCCGGAGGATCATTGAGACTGTCATGCGAAGCGAGCGGTTTTATCTTCTCCGATTACT >TE13-AT 19LDATGGGATGGATTCGGCAGGCCCCGGGAAAGGGACTCGAATGGGTGTCATACATCGGAAGGTCAGGCTCGTCCATGTACTA(M13)CGCAGACTCGGTGAAAGGCAGATTCACCTTTAGCCGGGACAACGCCAAGAATTCCCTCTACTTGCAGATGAACAGCCTGCGAGCCGAGGATACTGCTGTCTACTACTGTGCCGCGTCGCCGGTGGTGGCAGCTACTGAAGATTTCCAGCACTGGGGACAGGGAACTCTGGTCACGGTGTCGAGCGGTGGGGGCGGAAGCGGAGGCGGAGGATCGGGCGGCGGAGGTTCGGGGGGGGGAGGGTCTGACATCGTGATGACCCAAACCCCAGCCACCCTGAGCCTCTCCCCTGGAGAGCGCGCGACTCTTTCGTGCCGCGCTTCCCAGTCAGTGACCAGCAATTACTTGGCTTGGTACCAACAGAAGCCGGGACAGGCGCCACGGCTGCTGCTTTTTGGTGCCAGCACTCGCGCCACCGGAATCCCGGATCGCTTCTCGGGCTCAGGGTCCGGGACGGACTTCACCCTGACTATCAACCGGCTGGAACCTGAGGACTTCGCGATGTACTACTGCCAGCAGTACGGCTCCGCACCAGTCACTTTCGGACAAGGCACCAAGCTGGAGATCAAGACCACTACCCCAGCACCGAGGCCACCCACCCCGGCTCCTACCATCGCCTCCCAGCCTCTGTCCCTGCGTCCGGAGGCATGTAGACCCGCAGCTGGTGGGGCCGTGCATACCCGGGGTCTTGACTTCGCCTGCGATATCTACATTTGGGCCCCTCTGGCTGGTACTTGCGGGGTCCTGCTGCTTTCACTCGTGATCACTCTTTACTGTAAGCGCGGTCGGAAGAAGCTGCTGTACATCTTTAAGCAACCCTTCATGAGGCCTGTGCAGACTACTCAAGAGGAGGACGGCTGTTCATGCCGGTTCCCAGAGGAGGAGGAAGGCGGCTGCGAACTGCGCGTGAAATTCAGCCGCAGCGCAGATGCTCCAGCCTACAAGCAGGGGCAGAACCAGCTCTACAACGAACTCAATCTTGGTCGGAGAGAGGAGTACGACGTGCTGGACAAGCGGAGAGGACGGGACCCAGAAATGGGCGGGAAGCCGCGCAGAAAGAATCCCCAAGAGGGCCTGTACAACGAGCTCCAAAAGGATAAGATGGCAGAAGCCTATAGCGAGATTGGTATGAAAGGGGAACGCAGAAGAGGCAAAGGCCACGACGGACTGTACCAGGGACTCAGCACCGCCACCAAGGACACCTATGACGCTCTTCACATGCAGGCCCTGCCGCCTCGG 100 M14CAAGTCCAACTCGTCCAGTCGGGAGCAGAAGTTAGAGCACCAGGAGCGTCAGTGAAAATCTCATGCAAGGCCTCGGGCTT(ScFv CACGTTCCGCGGATACTAC domain)ATCCACTGGGTGCGCCAAGCCCCGGGTCAGGGATTGGAGTGGATGGGAATCATTAACCCATCAGGAGGGAGCCGGGCTTA >BS83-CGCGCAGAAGTTCCAGGGACGCGTCACTATGACCCGAGATACTTCCACCTCGACTGTGTACATGGAACTCTCGTCCCTGA95IDGGTCCGACGACACTGCGATGTATTACTGTGCTCGGACTGCCAGCTGCGGTGGGGACTGTTACTACCTCGATTACTGGGGC(M14)CAGGGAACTCTGGTGACCGTGTCCAGCGGAGGTGGCGGGTCAGGGGGTGGCGGAAGCGGAGGCGGCGGTTCAGGCGGAGGAGGCTCGGACATCCAAATGACGCAATCGCCGCCTACCCTGAGCGCTTCCGTGGGAGATCGGGTGACCATTACTTGCAGAGCATCCGAGAACGTCAATATCTGGCTGGCCTGGTACCAACAGAAGCCGGGGAAGGCCCCTAAACTGCTGATCTACAAGTCGAGCAGCCTTGCCTCTGGAGTGCCCTCCCGCTTCTCGGGCTCGGGATCAGGAGCGGAATTCACCCTCACCATCTCCTCCCTGCAGCCAGATGACTTTGCCACCTACTACTGCCAGCAGTACCAGAGCTATCCGTTGACCTTTGGGGGAGGCACTAAAGTGGACATCAAG 124 M14ATGGCCCTCCCTGTCACCGCCCTGCTGCTTCCGCTGGCTCTTCTGCTCCACGCCGCTCGGCCCCAAGTCCAACTCGTCCA(Full)GTCGGGAGCAGAAGTTAGAGCACCAGGAGCGTCAGTGAAAATCTCATGCAAGGCCTCGGGCTTCACGTTCCGCGGATACT >BS83-AC 95IDATCCACTGGGTGCGCCAAGCCCCGGGTCAGGGATTGGAGTGGATGGGAATCATTAACCCATCAGGAGGGAGCCGGGCTTA(M14)CGCGCAGAAGTTCCAGGGACGCGTCACTATGACCCGAGATACTTCCACCTCGACTGTGTACATGGAACTCTCGTCCCTGAGGTCCGACGACACTGCGATGTATTACTGTGCTCGGACTGCCAGCTGCGGTGGGGACTGTTACTACCTCGATTACTGGGGCCAGGGAACTCTGGTGACCGTGTCCAGCGGAGGTGGCGGGTCAGGGGGTGGCGGAAGCGGAGGCGGCGGTTCAGGCGGAGGAGGCTCGGACATCCAAATGACGCAATCGCCGCCTACCCTGAGCGCTTCCGTGGGAGATCGGGTGACCATTACTTGCAGAGCATCCGAGAACGTCAATATCTGGCTGGCCTGGTACCAACAGAAGCCGGGGAAGGCCCCTAAACTGCTGATCTACAAGTCGAGCAGCCTTGCCTCTGGAGTGCCCTCCCGCTTCTCGGGCTCGGGATCAGGAGCGGAATTCACCCTCACCATCTCCTCCCTGCAGCCAGATGACTTTGCCACCTACTACTGCCAGCAGTACCAGAGCTATCCGTTGACCTTTGGGGGAGGCACTAAAGTGGACATCAAGACCACTACCCCAGCACCGAGGCCACCCACCCCGGCTCCTACCATCGCCTCCCAGCCTCTGTCCCTGCGTCCGGAGGCATGTAGACCCGCAGCTGGTGGGGCCGTGCATACCCGGGGTCTTGACTTCGCCTGCGATATCTACATTTGGGCCCCTCTGGCTGGTACTTGCGGGGTCCTGCTGCTTTCACTCGTGATCACTCTTTACTGTAAGCGCGGTCGGAAGAAGCTGCTGTACATCTTTAAGCAACCCTTCATGAGGCCTGTGCAGACTACTCAAGAGGAGGACGGCTGTTCATGCCGGTTCCCAGAGGAGGAGGAAGGCGGCTGCGAACTGCGCGTGAAATTCAGCCGCAGCGCAGATGCTCCAGCCTACAAGCAGGGGCAGAACCAGCTCTACAACGAACTCAATCTTGGTCGGAGAGAGGAGTACGACGTGCTGGACAAGCGGAGAGGACGGGACCCAGAAATGGGCGGGAAGCCGCGCAGAAAGAATCCCCAAGAGGGCCTGTACAACGAGCTCCAAAAGGATAAGATGGCAGAAGCCTATAGCGAGATTGGTATGAAAGGGGAACGCAGAAGAGGCAAAGGCCACGACGGACTGTACCAGGGACTCAGCACCGCCACCAAGGACACCTATGACGCTCTTCACATGCAGGCCCTGCCGCCTCGG 101 M15 CAAGTTCAACTCGTTCAA (ScFvTCAGGTGGAGGACTCGTGCAACCAGGAAGATCACTCAGACTCAGCTGCGCCGCGTCGGGATTCACTTTCGATGACTACGCdomain)AATGCACTGGGTGCGGCAGGCCCCGGGCAAAGGACTGGAATGGGTGAGCGGAATTAGCTGGAACTCGGGGTCCATCGGGT >HS86-ACGCCGACTCGGTGAAGGGACGCTTTACGATCTCCCGGGACAATGCCAAGAACTCCCTGTATTTGCAGATGAACTCCTTG94XDAGGGCTGAGGACACCGCCGTGTACTACTGCGCTAAAGATGGATCATCGTCCTGGTCCTGGGGATACTTCGATTACTGGGG(M15)CCAGGGCACTCTGGTGACCGTGTCGTCAGGCGGTGGAGGGTCGGGCGGAGGAGGTAGCGGAGGCGGAGGGAGCAGCTCTGAACTGACCCAAGACCCGGCGGTGTCGGTCGCCCTTGGTCAGACTGTGCGGACTACCTGTCAGGGGGACGCGCTGCGCTCGTACTACGCTTCATGGTACCAGCAGAAGCCCGGACAGGCACCTATGCTGGTCATCTACGGAAAGAATAACCGCCCATCCGGCATCCCGGATCGCTTCTCGGGTTCGGACAGCGGCGACACCGCATCCCTGACGATCACTGGAGCGCAGGCCGAGGATGAAGCCGACTACTACTGCAATTCCCGAGATTCAAGCGGCTACCCTGTGTTTGGGACCGGAACTAAGGTCACCGTCCTG125 M15ATGGCCCTCCCTGTCACCGCCCTGCTGCTTCCGCTGGCTCTTCTGCTCCACGCCGCTCGGCCCCAAGTTCAACTCGTTCA(Full) A >HS86-TCAGGTGGAGGACTCGTGCAACCAGGAAGATCACTCAGACTCAGCTGCGCCGCGTCGGGATTCACTTTCGATGACTACGC94XDAATGCACTGGGTGCGGCAGGCCCCGGGCAAAGGACTGGAATGGGTGAGCGGAATTAGCTGGAACTCGGGGTCCATCGGGT(M15)ACGCCGACTCGGTGAAGGGACGCTTTACGATCTCCCGGGACAATGCCAAGAACTCCCTGTATTTGCAGATGAACTCCTTGAGGGCTGAGGACACCGCCGTGTACTACTGCGCTAAAGATGGATCATCGTCCTGGTCCTGGGGATACTTCGATTACTGGGGCCAGGGCACTCTGGTGACCGTGTCGTCAGGCGGTGGAGGGTCGGGCGGAGGAGGTAGCGGAGGCGGAGGGAGCAGCTCTGAACTGACCCAAGACCCGGCGGTGTCGGTCGCCCTTGGTCAGACTGTGCGGACTACCTGTCAGGGGGACGCGCTGCGCTCGTACTACGCTTCATGGTACCAGCAGAAGCCCGGACAGGCACCTATGCTGGTCATCTACGGAAAGAATAACCGCCCATCCGGCATCCCGGATCGCTTCTCGGGTTCGGACAGCGGCGACACCGCATCCCTGACGATCACTGGAGCGCAGGCCGAGGATGAAGCCGACTACTACTGCAATTCCCGAGATTCAAGCGGCTACCCTGTGTTTGGGACCGGAACTAAGGTCACCGTCCTGACCACTACCCCAGCACCGAGGCCACCCACCCCGGCTCCTACCATCGCCTCCCAGCCTCTGTCCCTGCGTCCGGAGGCATGTAGACCCGCAGCTGGTGGGGCCGTGCATACCCGGGGTCTTGACTTCGCCTGCGATATCTACATTTGGGCCCCTCTGGCTGGTACTTGCGGGGTCCTGCTGCTTTCACTCGTGATCACTCTTTACTGTAAGCGCGGTCGGAAGAAGCTGCTGTACATCTTTAAGCAACCCTTCATGAGGCCTGTGCAGACTACTCAAGAGGAGGACGGCTGTTCATGCCGGTTCCCAGAGGAGGAGGAAGGCGGCTGCGAACTGCGCGTGAAATTCAGCCGCAGCGCAGATGCTCCAGCCTACAAGCAGGGGCAGAACCAGCTCTACAACGAACTCAATCTTGGTCGGAGAGAGGAGTACGACGTGCTGGACAAGCGGAGAGGACGGGACCCAGAAATGGGCGGGAAGCCGCGCAGAAAGAATCCCCAAGAGGGCCTGTACAACGAGCTCCAAAAGGATAAGATGGCAGAAGCCTATAGCGAGATTGGTATGAAAGGGGAACGCAGAAGAGGCAAAGGCCACGACGGACTGTACCAGGGACTCAGCACCGCCACCAAGGACACCTATGACGCTCTTCACATGCAGGCCCTGCCGCCTCGG 102 M16 GAAGTGCAACTCGTGGAA (ScFvTCTGGTGGAGGACTTGTGCAACCTGGAAGATCGTTGAGACTCTCATGTGCTGCCTCCGGGTTCACCTTTGACGACTACGCdomain)CATGCACTGGGTGCGCCAGGCACCAGGAAAGGGTCTGGAGTGGGTTTCGGGTATCTCGTGGAACTCCGGGAGCACTGGCT >XS87-ACGCTGATTCGGTGAAAGGCCGGTTTACCATCTCCCGAGACAATGCGAAGAATTCCCTCTATCTGCAGATGAACAGCCTC99RDCGGGCCGAGGATACTGCCCTGTACTACTGCGCCAAGGATAGCTCATCATGGTACGGAGGTGGATCGGCTTTCGATATCTG(M16)GGGCCAGGGCACGATGGTCACCGTGTCCTCGGGGGGCGGAGGCTCCGGGGGAGGAGGTAGCGGAGGAGGAGGATCGAGCTCAGAGTTGACTCAAGAACCCGCAGTGTCCGTGGCACTGGGCCAAACCGTCAGGATCACTTGCCAGGGAGACAGCCTGAGGTCGTACTACGCGTCCTGGTACCAGCAGAAGCCGGGACAGGCCCCGGTCCTGGTCATTTTCGGACGCTCAAGACGCCCATCGGGCATCCCGGACCGGTTCAGCGGAAGCTCCTCGGGAAACACCGCGTCACTTATCATTACCGGCGCACAGGCTGAGGACGAAGCGGATTACTACTGCAACTCCCGCGACAATACTGCCAACCATTACGTGTTCGGGACCGGAACGAAACTGACTGTCCTG126 M16ATGGCCCTCCCTGTCACCGCCCTGCTGCTTCCGCTGGCTCTTCTGCTCCACGCCGCTCGGCCCGAAGTGCAACTCGTGGA(Full) A >XS87-TCTGGTGGAGGACTTGTGCAACCTGGAAGATCGTTGAGACTCTCATGTGCTGCCTCCGGGTTCACCTTTGACGACTACGC99RDCATGCACTGGGTGCGCCAGGCACCAGGAAAGGGTCTGGAGTGGGTTTCGGGTATCTCGTGGAACTCCGGGAGCACTGGCT(M16)ACGCTGATTCGGTGAAAGGCCGGTTTACCATCTCCCGAGACAATGCGAAGAATTCCCTCTATCTGCAGATGAACAGCCTCCGGGCCGAGGATACTGCCCTGTACTACTGCGCCAAGGATAGCTCATCATGGTACGGAGGTGGATCGGCTTTCGATATCTGGGGCCAGGGCACGATGGTCACCGTGTCCTCGGGGGGCGGAGGCTCCGGGGGAGGAGGTAGCGGAGGAGGAGGATCGAGCTCAGAGTTGACTCAAGAACCCGCAGTGTCCGTGGCACTGGGCCAAACCGTCAGGATCACTTGCCAGGGAGACAGCCTGAGGTCGTACTACGCGTCCTGGTACCAGCAGAAGCCGGGACAGGCCCCGGTCCTGGTCATTTTCGGACGCTCAAGACGCCCATCGGGCATCCCGGACCGGTTCAGCGGAAGCTCCTCGGGAAACACCGCGTCACTTATCATTACCGGCGCACAGGCTGAGGACGAAGCGGATTACTACTGCAACTCCCGCGACAATACTGCCAACCATTACGTGTTCGGGACCGGAACGAAACTGACTGTCCTGACCACTACCCCAGCACCGAGGCCACCCACCCCGGCTCCTACCATCGCCTCCCAGCCTCTGTCCCTGCGTCCGGAGGCATGTAGACCCGCAGCTGGTGGGGCCGTGCATACCCGGGGTCTTGACTTCGCCTGCGATATCTACATTTGGGCCCCTCTGGCTGGTACTTGCGGGGTCCTGCTGCTTTCACTCGTGATCACTCTTTACTGTAAGCGCGGTCGGAAGAAGCTGCTGTACATCTTTAAGCAACCCTTCATGAGGCCTGTGCAGACTACTCAAGAGGAGGACGGCTGTTCATGCCGGTTCCCAGAGGAGGAGGAAGGCGGCTGCGAACTGCGCGTGAAATTCAGCCGCAGCGCAGATGCTCCAGCCTACAAGCAGGGGCAGAACCAGCTCTACAACGAACTCAATCTTGGTCGGAGAGAGGAGTACGACGTGCTGGACAAGCGGAGAGGACGGGACCCAGAAATGGGCGGGAAGCCGCGCAGAAAGAATCCCCAAGAGGGCCTGTACAACGAGCTCCAAAAGGATAAGATGGCAGAAGCCTATAGCGAGATTGGTATGAAAGGGGAACGCAGAAGAGGCAAAGGCCACGACGGACTGTACCAGGGACTCAGCACCGCCACCAAGGACACCTATGACGCTCTTCACATGCAGGCCCTGCCGCCTCGG 103 M17 GAAGTTCAATTGGTGGAA (ScFvTCTGGAGGAGGACTTGTGCAACCCGGTAGATCTCTGAGACTGTCCTGTGCGGCATCGGGATTCACCTTCGACGACTACGCdomain)TATGCACTGGGTGAGACAAGCCCCTGGAAAAGGACTGGAGTGGGTGTCAGGCATCTCCTGGAATAGCGGGTCCACTGGAT >NS89-ACGCCGATTCGGTCAAGGGTCGCTTCACCATTTCCCGGGACAATGCCAAGAACTCCCTGTACCTTCAAATGAACTCCCTC94MDCGGGCCGAGGATACCGCCCTCTACTACTGCGCCAAAGACAGCTCGTCATGGTATGGCGGAGGGTCGGCATTTGACATCTG(M17)GGGACAGGGAACTATGGTGACTGTGTCATCAGGAGGCGGCGGAAGCGGCGGCGGCGGGTCCGGCGGAGGAGGGTCGTCCAGCGAACTCACCCAAGATCCAGCAGTGAGCGTCGCGCTGGGCCAGACCGTCAGGATCACGTGCCAGGGAGATTCACTGCGCTCATACTACGCGTCCTGGTACCAGCAGAAGCCGGGGCAGGCCCCGGTCCTCGTGATCTACGGAAAGAACAACCGCCCGTCGGGTATCCCAGACCGCTTTTCGGGTAGCTCCAGCGGAAATACGGCTAGCCTGACCATCACTGGAGCACAGGCTGAGGATGAAGCGGACTACTACTGCAATTCGCGGGGCTCATCGGGGAACCATTACGTGTTCGGAACTGGTACCAAGGTGACTGTCCTG127 M17ATGGCCCTCCCTGTCACCGCCCTGCTGCTTCCGCTGGCTCTTCTGCTCCACGCCGCTCGGCCCGAAGTTCAATTGGTGGA(Full) A >NS89-TCTGGAGGAGGACTTGTGCAACCCGGTAGATCTCTGAGACTGTCCTGTGCGGCATCGGGATTCACCTTCGACGACTACGC94MDTATGCACTGGGTGAGACAAGCCCCTGGAAAAGGACTGGAGTGGGTGTCAGGCATCTCCTGGAATAGCGGGTCCACTGGAT(M17)ACGCCGATTCGGTCAAGGGTCGCTTCACCATTTCCCGGGACAATGCCAAGAACTCCCTGTACCTTCAAATGAACTCCCTCCGGGCCGAGGATACCGCCCTCTACTACTGCGCCAAAGACAGCTCGTCATGGTATGGCGGAGGGTCGGCATTTGACATCTGGGGACAGGGAACTATGGTGACTGTGTCATCAGGAGGCGGCGGAAGCGGCGGCGGCGGGTCCGGCGGAGGAGGGTCGTCCAGCGAACTCACCCAAGATCCAGCAGTGAGCGTCGCGCTGGGCCAGACCGTCAGGATCACGTGCCAGGGAGATTCACTGCGCTCATACTACGCGTCCTGGTACCAGCAGAAGCCGGGGCAGGCCCCGGTCCTCGTGATCTACGGAAAGAACAACCGCCCGTCGGGTATCCCAGACCGCTTTTCGGGTAGCTCCAGCGGAAATACGGCTAGCCTGACCATCACTGGAGCACAGGCTGAGGATGAAGCGGACTACTACTGCAATTCGCGGGGCTCATCGGGGAACCATTACGTGTTCGGAACTGGTACCAAGGTGACTGTCCTGACCACTACCCCAGCACCGAGGCCACCCACCCCGGCTCCTACCATCGCCTCCCAGCCTCTGTCCCTGCGTCCGGAGGCATGTAGACCCGCAGCTGGTGGGGCCGTGCATACCCGGGGTCTTGACTTCGCCTGCGATATCTACATTTGGGCCCCTCTGGCTGGTACTTGCGGGGTCCTGCTGCTTTCACTCGTGATCACTCTTTACTGTAAGCGCGGTCGGAAGAAGCTGCTGTACATCTTTAAGCAACCCTTCATGAGGCCTGTGCAGACTACTCAAGAGGAGGACGGCTGTTCATGCCGGTTCCCAGAGGAGGAGGAAGGCGGCTGCGAACTGCGCGTGAAATTCAGCCGCAGCGCAGATGCTCCAGCCTACAAGCAGGGGCAGAACCAGCTCTACAACGAACTCAATCTTGGTCGGAGAGAGGAGTACGACGTGCTGGACAAGCGGAGAGGACGGGACCCAGAAATGGGCGGGAAGCCGCGCAGAAAGAATCCCCAAGAGGGCCTGTACAACGAGCTCCAAAAGGATAAGATGGCAGAAGCCTATAGCGAGATTGGTATGAAAGGGGAACGCAGAAGAGGCAAAGGCCACGACGGACTGTACCAGGGACTCAGCACCGCCACCAAGGACACCTATGACGCTCTTCACATGCAGGCCCTGCCGCCTCGG 104 M18CAAGTGCAGCTCGTTCAATCAGGCGGAGGACTCGTTCAACCAGGAGGATCATTGCGACTCTCATGTGCGGCCTCTGGATT(ScFv CACGTTTAGCTCATATTGG domain)ATGCACTGGGTGCGGCAGGCGCCGGGGAAAGGTCTGGTGTGGGTCAGCCGCATCAACTCAGACGGCTCCTCGACTTCGTA >DS90-CGCCGACTCCGTGAAGGGACGCTTTACCATTTCCCGCGACAACGCCAAGAATACCCTTTACCTTCAGATGAACTCCCTCC09HDGCGCTGAGGATACCGCCGTGTACTACTGCGTGAGGACTGGCTGGGTCGGCAGCTACTACTACTACATGGACGTGTGGGGC(M18)AAAGGAACTACTGTCACCGTGTCAAGCGGCGGTGGAGGTTCCGGCGGGGGAGGATCGGGGGGGGGCGGATCGGGTGGCGGAGGATCGGAGATCGTGTTGACCCAGTCGCCGGGAACCCTGTCGCTGTCGCCTGGGGAGAGAGCAACTCTGTCCTGCCGGGCTTCCCAGTCGGTGTCGAGCAATTACCTGGCATGGTACCAACAGAAGCCGGGACAGCCGCCACGCCTGCTGATCTATGACGTGTCAACTCGGGCAACTGGAATCCCTGCGCGGTTCAGCGGCGGAGGGAGCGGTACCGATTTCACCCTGACTATTTCCTCCCTCGAACCAGAAGATTTCGCCGTCTACTACTGCCAGCAGAGAAGCAACTGGCCGCCCTGGACGTTCGGACAAGGAACCAAGGTCGAAATCAAG 128 M18ATGGCCCTCCCTGTCACCGCCCTGCTGCTTCCGCTGGCTCTTCTGCTCCACGCCGCTCGGCCCCAAGTGCAGCTCGTTCA(Full)ATCAGGCGGAGGACTCGTTCAACCAGGAGGATCATTGCGACTCTCATGTGCGGCCTCTGGATTCACGTTTAGCTCATATT >DS90-GG 09HDATGCACTGGGTGCGGCAGGCGCCGGGGAAAGGTCTGGTGTGGGTCAGCCGCATCAACTCAGACGGCTCCTCGACTTCGTA(M18)CGCCGACTCCGTGAAGGGACGCTTTACCATTTCCCGCGACAACGCCAAGAATACCCTTTACCTTCAGATGAACTCCCTCCGCGCTGAGGATACCGCCGTGTACTACTGCGTGAGGACTGGCTGGGTCGGCAGCTACTACTACTACATGGACGTGTGGGGCAAAGGAACTACTGTCACCGTGTCAAGCGGCGGTGGAGGTTCCGGCGGGGGAGGATCGGGGGGGGGCGGATCGGGTGGCGGAGGATCGGAGATCGTGTTGACCCAGTCGCCGGGAACCCTGTCGCTGTCGCCTGGGGAGAGAGCAACTCTGTCCTGCCGGGCTTCCCAGTCGGTGTCGAGCAATTACCTGGCATGGTACCAACAGAAGCCGGGACAGCCGCCACGCCTGCTGATCTATGACGTGTCAACTCGGGCAACTGGAATCCCTGCGCGGTTCAGCGGCGGAGGGAGCGGTACCGATTTCACCCTGACTATTTCCTCCCTCGAACCAGAAGATTTCGCCGTCTACTACTGCCAGCAGAGAAGCAACTGGCCGCCCTGGACGTTCGGACAAGGAACCAAGGTCGAAATCAAGACCACTACCCCAGCACCGAGGCCACCCACCCCGGCTCCTACCATCGCCTCCCAGCCTCTGTCCCTGCGTCCGGAGGCATGTAGACCCGCAGCTGGTGGGGCCGTGCATACCCGGGGTCTTGACTTCGCCTGCGATATCTACATTTGGGCCCCTCTGGCTGGTACTTGCGGGGTCCTGCTGCTTTCACTCGTGATCACTCTTTACTGTAAGCGCGGTCGGAAGAAGCTGCTGTACATCTTTAAGCAACCCTTCATGAGGCCTGTGCAGACTACTCAAGAGGAGGACGGCTGTTCATGCCGGTTCCCAGAGGAGGAGGAAGGCGGCTGCGAACTGCGCGTGAAATTCAGCCGCAGCGCAGATGCTCCAGCCTACAAGCAGGGGCAGAACCAGCTCTACAACGAACTCAATCTTGGTCGGAGAGAGGAGTACGACGTGCTGGACAAGCGGAGAGGACGGGACCCAGAAATGGGCGGGAAGCCGCGCAGAAAGAATCCCCAAGAGGGCCTGTACAACGAGCTCCAAAAGGATAAGATGGCAGAAGCCTATAGCGAGATTGGTATGAAAGGGGAACGCAGAAGAGGCAAAGGCCACGACGGACTGTACCAGGGACTCAGCACCGCCACCAAGGACACCTATGACGCTCTTCACATGCAGGCCCTGCCGCCTCGG 105 M19 CAAGTGCAATTGGTTCAA(ScFvTCAGGAGGAGGAGTCGTGCAGCCCGGAAGATCGTTGAGACTGTCATGTGCCGCGAGCGGCTTTACTTTCTCAAGCTACGGdomain)AATGCATTGGGTGCGACAGGCTCCGGGAAAAGGACTGGAATGGGTCGCAGTGATCTCATACGACGGCTCGAACAAGTACT >TS92-ACGCCGACTCCGTCAAGGGTCGGTTCACGATTTCGCGCGATAATTCCAAGAACACTCTGTACCTCCAAATGAACAGCCTC04BDCGGGCAGAGGACACCGCCGTCTACTACTGCGCTAAGGGATACTCGCGCTACTACTACTATGGAATGGATGTGTGGGGCCA(M19)GGGAACTACCGTGACGGTGTCGTCCGGCGGCGGTGGGTCGGGCGGAGGCGGATCAGGTGGAGGTGGAAGCGGAGGAGGAGGGAGCGAAATCGTCATGACTCAGTCCCCTGCTACCCTTTCTCTGTCGCCGGGAGAAAGAGCCATCCTGAGCTGCCGGGCCTCCCAGAGCGTGTACACCAAATACCTGGGATGGTACCAGCAGAAGCCGGGGCAGGCACCAAGGCTCCTGATCTACGATGCGTCCACCCGCGCGACTGGTATCCCAGACCGCTTTTCCGGCTCGGGGTCAGGGACTGACTTCACCCTTACTATCAATCGGCTCGAGCCTGAGGATTTCGCCGTGTATTACTGCCAGCACTACGGAGGGTCCCCGCTGATTACCTTCGGCCAAGGCACCAAAGTGGACATCAAG 129 M19ATGGCCCTCCCTGTCACCGCCCTGCTGCTTCCGCTGGCTCTTCTGCTCCACGCCGCTCGGCCCCAAGTGCAATTGGTTCA(Full) A >TS92-TCAGGAGGAGGAGTCGTGCAGCCCGGAAGATCGTTGAGACTGTCATGTGCCGCGAGCGGCTTTACTTTCTCAAGCTACGG04BDAATGCATTGGGTGCGACAGGCTCCGGGAAAAGGACTGGAATGGGTCGCAGTGATCTCATACGACGGCTCGAACAAGTACT(M19)ACGCCGACTCCGTCAAGGGTCGGTTCACGATTTCGCGCGATAATTCCAAGAACACTCTGTACCTCCAAATGAACAGCCTCCGGGCAGAGGACACCGCCGTCTACTACTGCGCTAAGGGATACTCGCGCTACTACTACTATGGAATGGATGTGTGGGGCCAGGGAACTACCGTGACGGTGTCGTCCGGCGGCGGTGGGTCGGGCGGAGGCGGATCAGGTGGAGGTGGAAGCGGAGGAGGAGGGAGCGAAATCGTCATGACTCAGTCCCCTGCTACCCTTTCTCTGTCGCCGGGAGAAAGAGCCATCCTGAGCTGCCGGGCCTCCCAGAGCGTGTACACCAAATACCTGGGATGGTACCAGCAGAAGCCGGGGCAGGCACCAAGGCTCCTGATCTACGATGCGTCCACCCGCGCGACTGGTATCCCAGACCGCTTTTCCGGCTCGGGGTCAGGGACTGACTTCACCCTTACTATCAATCGGCTCGAGCCTGAGGATTTCGCCGTGTATTACTGCCAGCACTACGGAGGGTCCCCGCTGATTACCTTCGGCCAAGGCACCAAAGTGGACATCAAGACCACTACCCCAGCACCGAGGCCACCCACCCCGGCTCCTACCATCGCCTCCCAGCCTCTGTCCCTGCGTCCGGAGGCATGTAGACCCGCAGCTGGTGGGGCCGTGCATACCCGGGGTCTTGACTTCGCCTGCGATATCTACATTTGGGCCCCTCTGGCTGGTACTTGCGGGGTCCTGCTGCTTTCACTCGTGATCACTCTTTACTGTAAGCGCGGTCGGAAGAAGCTGCTGTACATCTTTAAGCAACCCTTCATGAGGCCTGTGCAGACTACTCAAGAGGAGGACGGCTGTTCATGCCGGTTCCCAGAGGAGGAGGAAGGCGGCTGCGAACTGCGCGTGAAATTCAGCCGCAGCGCAGATGCTCCAGCCTACAAGCAGGGGCAGAACCAGCTCTACAACGAACTCAATCTTGGTCGGAGAGAGGAGTACGACGTGCTGGACAAGCGGAGAGGACGGGACCCAGAAATGGGCGGGAAGCCGCGCAGAAAGAATCCCCAAGAGGGCCTGTACAACGAGCTCCAAAAGGATAAGATGGCAGAAGCCTATAGCGAGATTGGTATGAAAGGGGAACGCAGAAGAGGCAAAGGCCACGACGGACTGTACCAGGGACTCAGCACCGCCACCAAGGACACCTATGACGCTCTTCACATGCAGGCCCTGCCGCCTCGG 106 M20CAAGTGCAACTTGTTCAATCAGGAGGAGGACTCGTTCAACCCGGAGGATCACTGCGACTCTCATGTGCAGCGTCGGGGTT(ScFv CACCTTCTCCAGCTACGCA domain)ATGTCCTGGGTGCGCCAAGCCCCTGGAAAAGGCCTGGAGTGGGTGTCGGCCATCTCTGGGAGCGGGGGATCAACTTACTA >JS93-CGCTGACTCCGTCAAGGGCCGCTTTACCATCTCCCGGGACAACAGCAAGAACACTCTCTATCTCCAGATGAACTCGCTGA08WDGAGCCGAAGATACCGCTGTCTACTACTGCGCGAAGAGAGAAGCTGCCGCAGGGCACGATTGGTACTTCGACTTGTGGGGC(M20)AGGGGCACCCTTGTGACCGTGTCCTCCGGTGGAGGCGGATCAGGAGGTGGGGGATCGGGTGGAGGAGGAAGCGGAGGCGGCGGTTCGGACATTCGCGTCACCCAGTCACCGAGCTCCCTCAGCGCATCGGTGGGCGACCGGGTCACTATCACTTGCCGGGCGTCCCAGTCGATCTCATCGTATCTGAATTGGTACCAGCAGAAACCGGGAAAGGCGCCGAAGCTGTTGATCTACGCTGCCAGCTCCCTGCAGTCGGGTGTGCCATCACGCTTTTCCGGCTCGGGATCGGGAACCGATTTCACTCTGACGATCTCTAGCCTGCAGCCAGAAGATTTCGCCACTTACTACTGCCAGCAGTCCTACAGCATCCCTCTGACTTTCGGACAAGGGACGAAAGTGGAGATTAAG 130 M20ATGGCCCTCCCTGTCACCGCCCTGCTGCTTCCGCTGGCTCTTCTGCTCCACGCCGCTCGGCCCCAAGTGCAACTTGTTCA(Full)ATCAGGAGGAGGACTCGTTCAACCCGGAGGATCACTGCGACTCTCATGTGCAGCGTCGGGGTTCACCTTCTCCAGCTACG >JS93-CA 08WDATGTCCTGGGTGCGCCAAGCCCCTGGAAAAGGCCTGGAGTGGGTGTCGGCCATCTCTGGGAGCGGGGGATCAACTTACTA(M20)CGCTGACTCCGTCAAGGGCCGCTTTACCATCTCCCGGGACAACAGCAAGAACACTCTCTATCTCCAGATGAACTCGCTGAGAGCCGAAGATACCGCTGTCTACTACTGCGCGAAGAGAGAAGCTGCCGCAGGGCACGATTGGTACTTCGACTTGTGGGGCAGGGGCACCCTTGTGACCGTGTCCTCCGGTGGAGGCGGATCAGGAGGTGGGGGATCGGGTGGAGGAGGAAGCGGAGGCGGCGGTTCGGACATTCGCGTCACCCAGTCACCGAGCTCCCTCAGCGCATCGGTGGGCGACCGGGTCACTATCACTTGCCGGGCGTCCCAGTCGATCTCATCGTATCTGAATTGGTACCAGCAGAAACCGGGAAAGGCGCCGAAGCTGTTGATCTACGCTGCCAGCTCCCTGCAGTCGGGTGTGCCATCACGCTTTTCCGGCTCGGGATCGGGAACCGATTTCACTCTGACGATCTCTAGCCTGCAGCCAGAAGATTTCGCCACTTACTACTGCCAGCAGTCCTACAGCATCCCTCTGACTTTCGGACAAGGGACGAAAGTGGAGATTAAGACCACTACCCCAGCACCGAGGCCACCCACCCCGGCTCCTACCATCGCCTCCCAGCCTCTGTCCCTGCGTCCGGAGGCATGTAGACCCGCAGCTGGTGGGGCCGTGCATACCCGGGGTCTTGACTTCGCCTGCGATATCTACATTTGGGCCCCTCTGGCTGGTACTTGCGGGGTCCTGCTGCTTTCACTCGTGATCACTCTTTACTGTAAGCGCGGTCGGAAGAAGCTGCTGTACATCTTTAAGCAACCCTTCATGAGGCCTGTGCAGACTACTCAAGAGGAGGACGGCTGTTCATGCCGGTTCCCAGAGGAGGAGGAAGGCGGCTGCGAACTGCGCGTGAAATTCAGCCGCAGCGCAGATGCTCCAGCCTACAAGCAGGGGCAGAACCAGCTCTACAACGAACTCAATCTTGGTCGGAGAGAGGAGTACGACGTGCTGGACAAGCGGAGAGGACGGGACCCAGAAATGGGCGGGAAGCCGCGCAGAAAGAATCCCCAAGAGGGCCTGTACAACGAGCTCCAAAAGGATAAGATGGCAGAAGCCTATAGCGAGATTGGTATGAAAGGGGAACGCAGAAGAGGCAAAGGCCACGACGGACTGTACCAGGGACTCAGCACCGCCACCAAGGACACCTATGACGCTCTTCACATGCAGGCCCTGCCGCCTCGG 107 M21CAAGTCCAACTCGTTCAGTCATGGGCAGAAGTCAAGAAACCCGGTGCAAGCGTCAAAGTGTCGTGTAAGGCCTCCGGCTA(ScFv CACTTTCACTTCCTACTAC domain)ATGCACTGGGTGCGCCAAGCCCCGGGACAGGGCCTTGAATGGATGGGCATCATCAACCCATCAGGAGGTTCCACGAGCTA >ZS95-CGCGCAGAAGTTCCAGGGGAGAGTGACGATGACTAGAGATACCTCCACGAGCACCGTCTACATGGAGCTGTCGAATCTGC03QDGGTCAGAGGACACTGCTGTGTATTACTGCGCGCGCTCCCCGCGGGTGACCACTGGCTACTTTGACTACTGGGGACAAGGG(M21)ACCCTGGTGACCGTCAGCTCGGGAGGCGGAGGATCGGGAGGTGGAGGGTCCGGTGGAGGCGGCTCTGGAGGAGGCGGGTCGGACATTCAATTGACCCAGAGCCCATCCACCCTCTCAGCCTCGGTGGGGGATAGGGTGACTATCACTTGCCGGGCCTCCCAGTCAATTTCCAGCTGGCTGGCTTGGTACCAGCAAAAGCCTGGAAAGGCACCGAAGCTCCTGATCTACAAGGCCTCATCTCTGGAATCAGGAGTGCCTTCGCGCTTCAGCGGAAGCGGCTCGGGAACTGAGTTTACCCTGACCATCTCGAGCCTGCAGCCAGATGACTTCGCGACCTATTACTGCCAGCAGTACTCGTCCTACCCGTTGACTTTCGGAGGAGGTACCCGCCTCGAAATCAAA 131 M21ATGGCCCTCCCTGTCACCGCCCTGCTGCTTCCGCTGGCTCTTCTGCTCCACGCCGCTCGGCCCCAAGTCCAACTCGTTCA(Full)GTCATGGGCAGAAGTCAAGAAACCCGGTGCAAGCGTCAAAGTGTCGTGTAAGGCCTCCGGCTACACTTTCACTTCCTACT >ZS95-AC 03QDATGCACTGGGTGCGCCAAGCCCCGGGACAGGGCCTTGAATGGATGGGCATCATCAACCCATCAGGAGGTTCCACGAGCTA(M21)CGCGCAGAAGTTCCAGGGGAGAGTGACGATGACTAGAGATACCTCCACGAGCACCGTCTACATGGAGCTGTCGAATCTGCGGTCAGAGGACACTGCTGTGTATTACTGCGCGCGCTCCCCGCGGGTGACCACTGGCTACTTTGACTACTGGGGACAAGGGACCCTGGTGACCGTCAGCTCGGGAGGCGGAGGATCGGGAGGTGGAGGGTCCGGTGGAGGCGGCTCTGGAGGAGGCGGGTCGGACATTCAATTGACCCAGAGCCCATCCACCCTCTCAGCCTCGGTGGGGGATAGGGTGACTATCACTTGCCGGGCCTCCCAGTCAATTTCCAGCTGGCTGGCTTGGTACCAGCAAAAGCCTGGAAAGGCACCGAAGCTCCTGATCTACAAGGCCTCATCTCTGGAATCAGGAGTGCCTTCGCGCTTCAGCGGAAGCGGCTCGGGAACTGAGTTTACCCTGACCATCTCGAGCCTGCAGCCAGATGACTTCGCGACCTATTACTGCCAGCAGTACTCGTCCTACCCGTTGACTTTCGGAGGAGGTACCCGCCTCGAAATCAAAACCACTACCCCAGCACCGAGGCCACCCACCCCGGCTCCTACCATCGCCTCCCAGCCTCTGTCCCTGCGTCCGGAGGCATGTAGACCCGCAGCTGGTGGGGCCGTGCATACCCGGGGTCTTGACTTCGCCTGCGATATCTACATTTGGGCCCCTCTGGCTGGTACTTGCGGGGTCCTGCTGCTTTCACTCGTGATCACTCTTTACTGTAAGCGCGGTCGGAAGAAGCTGCTGTACATCTTTAAGCAACCCTTCATGAGGCCTGTGCAGACTACTCAAGAGGAGGACGGCTGTTCATGCCGGTTCCCAGAGGAGGAGGAAGGCGGCTGCGAACTGCGCGTGAAATTCAGCCGCAGCGCAGATGCTCCAGCCTACAAGCAGGGGCAGAACCAGCTCTACAACGAACTCAATCTTGGTCGGAGAGAGGAGTACGACGTGCTGGACAAGCGGAGAGGACGGGACCCAGAAATGGGCGGGAAGCCGCGCAGAAAGAATCCCCAAGAGGGCCTGTACAACGAGCTCCAAAAGGATAAGATGGCAGAAGCCTATAGCGAGATTGGTATGAAAGGGGAACGCAGAAGAGGCAAAGGCCACGACGGACTGTACCAGGGACTCAGCACCGCCACCAAGGACACCTATGACGCTCTTCACATGCAGGCCCTGCCGCCTCGG 108 M22CAAGTCCAACTCGTCCAGTCCGGTGCAGAAGTCAGAAGGCCAGGAGCAAGCGTGAAGATCTCGTGTAGAGCGTCAGGAGA(ScFv CACCAGCACTCGCCATTAC domain)ATCCACTGGCTGCGCCAGGCTCCGGGCCAAGGGCCGGAGTGGATGGGTGTGATCAACCCGACTACGGGACCGGCTACCGG >PS96-AAGCCCTGCGTACGCACAGATGCTGCAGGGACGGGTGACTATGACCCGCGATACTAGCACTAGGACCGTGTACATGGAAC08LDTCCGCTCGTTGCGGTTCGAAGATACCGCCGTCTACTACTGCGCCCGGTCCGTGGTGGGCCGAAGCGCCCCTTACTACTTC(M22)GATTACTGGGGACAGGGCACTCTGGTGACCGTTAGCTCCGGTGGGGGAGGCTCGGGTGGAGGCGGATCGGGAGGAGGAGGCAGCGGTGGAGGGGGATCGGACATTCAGATGACCCAGTCACCCTCCTCCCTCTCAGCCTCGGTCGGGGACCGGGTGACCATTACGTGCAGAGCCTCACAAGGGATCTCGGACTACTCCGCCTGGTACCAGCAGAAACCGGGAAAAGCGCCAAAGCTCCTGATCTACGCCGCGAGCACCCTGCAATCAGGAGTGCCATCGCGCTTTTCTGGATCGGGCTCAGGGACTGACTTCACGCTGACTATCTCCTACCTTCAGTCCGAGGATTTCGCTACCTACTACTGCCAACAGTATTACTCCTATCCCCTGACCTTTGGCGGAGGCACTAAGGTGGACATCAAG 132 M22ATGGCCCTCCCTGTCACCGCCCTGCTGCTTCCGCTGGCTCTTCTGCTCCACGCCGCTCGGCCCCAAGTCCAACTCGTCCA(Full)GTCCGGTGCAGAAGTCAGAAGGCCAGGAGCAAGCGTGAAGATCTCGTGTAGAGCGTCAGGAGACACCAGCACTCGCCATT >PS96-AC 08LDATCCACTGGCTGCGCCAGGCTCCGGGCCAAGGGCCGGAGTGGATGGGTGTGATCAACCCGACTACGGGACCGGCTACCGG(M22)AAGCCCTGCGTACGCACAGATGCTGCAGGGACGGGTGACTATGACCCGCGATACTAGCACTAGGACCGTGTACATGGAACTCCGCTCGTTGCGGTTCGAAGATACCGCCGTCTACTACTGCGCCCGGTCCGTGGTGGGCCGAAGCGCCCCTTACTACTTCGATTACTGGGGACAGGGCACTCTGGTGACCGTTAGCTCCGGTGGGGGAGGCTCGGGTGGAGGCGGATCGGGAGGAGGAGGCAGCGGTGGAGGGGGATCGGACATTCAGATGACCCAGTCACCCTCCTCCCTCTCAGCCTCGGTCGGGGACCGGGTGACCATTACGTGCAGAGCCTCACAAGGGATCTCGGACTACTCCGCCTGGTACCAGCAGAAACCGGGAAAAGCGCCAAAGCTCCTGATCTACGCCGCGAGCACCCTGCAATCAGGAGTGCCATCGCGCTTTTCTGGATCGGGCTCAGGGACTGACTTCACGCTGACTATCTCCTACCTTCAGTCCGAGGATTTCGCTACCTACTACTGCCAACAGTATTACTCCTATCCCCTGACCTTTGGCGGAGGCACTAAGGTGGACATCAAGACCACTACCCCAGCACCGAGGCCACCCACCCCGGCTCCTACCATCGCCTCCCAGCCTCTGTCCCTGCGTCCGGAGGCATGTAGACCCGCAGCTGGTGGGGCCGTGCATACCCGGGGTCTTGACTTCGCCTGCGATATCTACATTTGGGCCCCTCTGGCTGGTACTTGCGGGGTCCTGCTGCTTTCACTCGTGATCACTCTTTACTGTAAGCGCGGTCGGAAGAAGCTGCTGTACATCTTTAAGCAACCCTTCATGAGGCCTGTGCAGACTACTCAAGAGGAGGACGGCTGTTCATGCCGGTTCCCAGAGGAGGAGGAAGGCGGCTGCGAACTGCGCGTGAAATTCAGCCGCAGCGCAGATGCTCCAGCCTACAAGCAGGGGCAGAACCAGCTCTACAACGAACTCAATCTTGGTCGGAGAGAGGAGTACGACGTGCTGGACAAGCGGAGAGGACGGGACCCAGAAATGGGCGGGAAGCCGCGCAGAAAGAATCCCCAAGAGGGCCTGTACAACGAGCTCCAAAAGGATAAGATGGCAGAAGCCTATAGCGAGATTGGTATGAAAGGGGAACGCAGAAGAGGCAAAGGCCACGACGGACTGTACCAGGGACTCAGCACCGCCACCAAGGACACCTATGACGCTCTTCACATGCAGGCCCTGCCGCCTCGG 109 M23CAAGTCCAACTCCAGCAATCGGGAGCAGAAGTCAAGAAACCAGGCGCATCGGTGAAAGTGTCGTGTAAGGCGTCAGGGTA(ScFv CACCTTCACCAACTACTAT domain)ATGCACTGGGTGCGCCAGGCTCCAGGCCAGGGGTTGGAGTGGATGGGGATCATCAATCCGTCAGGTGGCTACACCACTTA >XH66-CGCTCAGAAGTTCCAGGGACGCCTCACTATGACTCGCGATACTAGCACCTCCACGGTGTACATGGAACTGTCATCGCTGA84HEGGTCCGAAGATACCGCCGTCTACTACTGCGCACGGATCAGATCCTGCGGAGGAGATTGTTACTACTTTGACAACTGGGGA(M23)CAGGGCACCCTTGTTACTGTGTCATCGGGAGGAGGGGGAAGCGGAGGAGGTGGATCAGGCGGCGGTGGCAGCGGGGGCGGAGGATCGGACATTCAGCTGACTCAGTCCCCCTCCACTTTGTCGGCCAGCGTGGGAGACAGAGTGACCATCACTTGCCGGGCGTCCGAGAACGTCAATATCTGGCTGGCCTGGTACCAGCAAAAGCCTGGAAAAGCCCCGAAGCTGCTCATCTATAAGTCATCCAGCCTGGCGTCTGGTGTGCCGTCGCGGTTCTCCGGCAGCGGGAGCGGAGCCGAGTTCACTCTCACCATTTCGAGCCTTCAACCGGACGATTTCGCCACCTACTACTGCCAGCAGTACCAATCCTACCCTCTGACGTTTGGAGGTGGAACCAAGGTGGACATCAAG 133 M23ATGGCCCTCCCTGTCACCGCCCTGCTGCTTCCGCTGGCTCTTCTGCTCCACGCCGCTCGGCCCCAAGTCCAACTCCAGCA(Full)ATCGGGAGCAGAAGTCAAGAAACCAGGCGCATCGGTGAAAGTGTCGTGTAAGGCGTCAGGGTACACCTTCACCAACTACT >XH66-AT 84HEATGCACTGGGTGCGCCAGGCTCCAGGCCAGGGGTTGGAGTGGATGGGGATCATCAATCCGTCAGGTGGCTACACCACTTA(M23)CGCTCAGAAGTTCCAGGGACGCCTCACTATGACTCGCGATACTAGCACCTCCACGGTGTACATGGAACTGTCATCGCTGAGGTCCGAAGATACCGCCGTCTACTACTGCGCACGGATCAGATCCTGCGGAGGAGATTGTTACTACTTTGACAACTGGGGACAGGGCACCCTTGTTACTGTGTCATCGGGAGGAGGGGGAAGCGGAGGAGGTGGATCAGGCGGCGGTGGCAGCGGGGGCGGAGGATCGGACATTCAGCTGACTCAGTCCCCCTCCACTTTGTCGGCCAGCGTGGGAGACAGAGTGACCATCACTTGCCGGGCGTCCGAGAACGTCAATATCTGGCTGGCCTGGTACCAGCAAAAGCCTGGAAAAGCCCCGAAGCTGCTCATCTATAAGTCATCCAGCCTGGCGTCTGGTGTGCCGTCGCGGTTCTCCGGCAGCGGGAGCGGAGCCGAGTTCACTCTCACCATTTCGAGCCTTCAACCGGACGATTTCGCCACCTACTACTGCCAGCAGTACCAATCCTACCCTCTGACGTTTGGAGGTGGAACCAAGGTGGACATCAAGACCACTACCCCAGCACCGAGGCCACCCACCCCGGCTCCTACCATCGCCTCCCAGCCTCTGTCCCTGCGTCCGGAGGCATGTAGACCCGCAGCTGGTGGGGCCGTGCATACCCGGGGTCTTGACTTCGCCTGCGATATCTACATTTGGGCCCCTCTGGCTGGTACTTGCGGGGTCCTGCTGCTTTCACTCGTGATCACTCTTTACTGTAAGCGCGGTCGGAAGAAGCTGCTGTACATCTTTAAGCAACCCTTCATGAGGCCTGTGCAGACTACTCAAGAGGAGGACGGCTGTTCATGCCGGTTCCCAGAGGAGGAGGAAGGCGGCTGCGAACTGCGCGTGAAATTCAGCCGCAGCGCAGATGCTCCAGCCTACAAGCAGGGGCAGAACCAGCTCTACAACGAACTCAATCTTGGTCGGAGAGAGGAGTACGACGTGCTGGACAAGCGGAGAGGACGGGACCCAGAAATGGGCGGGAAGCCGCGCAGAAAGAATCCCCAAGAGGGCCTGTACAACGAGCTCCAAAAGGATAAGATGGCAGAAGCCTATAGCGAGATTGGTATGAAAGGGGAACGCAGAAGAGGCAAAGGCCACGACGGACTGTACCAGGGACTCAGCACCGCCACCAAGGACACCTATGACGCTCTTCACATGCAGGCCCTGCCGCCTCGG 110 M24 CAAATCACTCTGAAAGAA (ScFvTCTGGACCGGCCCTGGTTAAGCCGACTCAAACGCTCACCCTTACTTGCACCTTCAGCGGATTCTCACTCAGCACTGCTGGdomain)TGTGCACGTCGGATGGATTAGACAGCCGCCTGGAAAGGCCCTGGAATGGCTCGCCCTCATCTCCTGGGCCGATGACAAGA >NH67-GATACAGGCCCTCGCTTCGATCCCGGTTGGACATTACCCGGGTGACCTCGAAAGATCAGGTGGTGCTCTCAATGACCAAT89CEATGCAGCCGGAGGACACCGCTACGTACTACTGCGCACTGCAAGGATTTGACGGCTACGAGGCTAACTGGGGACCAGGTAC(M24)TCTGGTCACCGTGAGCTCCGGCGGGGGAGGATCAGGCGGGGGGGGGTCAGGAGGCGGAGGCTCCGGTGGAGGAGGATCGGATATCGTCATGACCCAGTCCCCAAGCTCGCTGAGCGCGTCAGCGGGCGACCGCGTGACTATCACTTGCCGGGCCAGCCGCGGCATCTCCTCCGCACTGGCGTGGTACCAGCAGAAGCCTGGAAAACCGCCAAAGCTCCTGATCTATGATGCCTCCAGCCTGGAGTCAGGTGTCCCCAGCCGCTTCTCGGGTTCGGGCTCGGGAACCGACTTCACTTTGACCATCGACTCGCTGGAACCGGAAGATTTCGCAACCTACTACTGTCAGCAGTCCTACTCGACCCCTTGGACTTTTGGACAAGGGACGAAGGTGGACATCAAG134 M24ATGGCCCTCCCTGTCACCGCCCTGCTGCTTCCGCTGGCTCTTCTGCTCCACGCCGCTCGGCCCCAAATCACTCTGAAAGA(Full) A >NH67-TCTGGACCGGCCCTGGTTAAGCCGACTCAAACGCTCACCCTTACTTGCACCTTCAGCGGATTCTCACTCAGCACTGCTGG89CETGTGCACGTCGGATGGATTAGACAGCCGCCTGGAAAGGCCCTGGAATGGCTCGCCCTCATCTCCTGGGCCGATGACAAGA(M24)GATACAGGCCCTCGCTTCGATCCCGGTTGGACATTACCCGGGTGACCTCGAAAGATCAGGTGGTGCTCTCAATGACCAATATGCAGCCGGAGGACACCGCTACGTACTACTGCGCACTGCAAGGATTTGACGGCTACGAGGCTAACTGGGGACCAGGTACTCTGGTCACCGTGAGCTCCGGCGGGGGAGGATCAGGCGGGGGGGGGTCAGGAGGCGGAGGCTCCGGTGGAGGAGGATCGGATATCGTCATGACCCAGTCCCCAAGCTCGCTGAGCGCGTCAGCGGGCGACCGCGTGACTATCACTTGCCGGGCCAGCCGCGGCATCTCCTCCGCACTGGCGTGGTACCAGCAGAAGCCTGGAAAACCGCCAAAGCTCCTGATCTATGATGCCTCCAGCCTGGAGTCAGGTGTCCCCAGCCGCTTCTCGGGTTCGGGCTCGGGAACCGACTTCACTTTGACCATCGACTCGCTGGAACCGGAAGATTTCGCAACCTACTACTGTCAGCAGTCCTACTCGACCCCTTGGACTTTTGGACAAGGGACGAAGGTGGACATCAAGACCACTACCCCAGCACCGAGGCCACCCACCCCGGCTCCTACCATCGCCTCCCAGCCTCTGTCCCTGCGTCCGGAGGCATGTAGACCCGCAGCTGGTGGGGCCGTGCATACCCGGGGTCTTGACTTCGCCTGCGATATCTACATTTGGGCCCCTCTGGCTGGTACTTGCGGGGTCCTGCTGCTTTCACTCGTGATCACTCTTTACTGTAAGCGCGGTCGGAAGAAGCTGCTGTACATCTTTAAGCAACCCTTCATGAGGCCTGTGCAGACTACTCAAGAGGAGGACGGCTGTTCATGCCGGTTCCCAGAGGAGGAGGAAGGCGGCTGCGAACTGCGCGTGAAATTCAGCCGCAGCGCAGATGCTCCAGCCTACAAGCAGGGGCAGAACCAGCTCTACAACGAACTCAATCTTGGTCGGAGAGAGGAGTACGACGTGCTGGACAAGCGGAGAGGACGGGACCCAGAAATGGGCGGGAAGCCGCGCAGAAAGAATCCCCAAGAGGGCCTGTACAACGAGCTCCAAAAGGATAAGATGGCAGAAGCCTATAGCGAGATTGGTATGAAAGGGGAACGCAGAAGAGGCAAAGGCCACGACGGACTGTACCAGGGACTCAGCACCGCCACCAAGGACACCTATGACGCTCTTCACATGCAGGCCCTGCCGCCTCGG 392 Ss1CAAGTCCAGCTCCAGCAGTCGGGCCCAGAGTTGGAGAAGCCTGGGGCGAGCGTGAAGAT (scFvCTCATGCAAAGCCTCAGGCTACTCCTTTACTGGATACACGATGAATTGGGTGAAACAGT domain)CGCATGGAAAGTCACTGGAATGGATCGGTCTGATTACGCCCTACAACGGCGCCTCCAGCTACAACCAGAAGTTCAGGGGAAAGGCGACCCTTACTGTCGACAAGTCGTCAAGCACCGCCTACATGGACCTCCTGTCCCTGACCTCCGAAGATAGCGCGGTCTACTTTTGTGCACGCGGAGGTTACGATGGACGGGGATTCGACTACTGGGGCCAGGGAACCACTGTCACCGTGTCGAGCGGAGGCGGAGGGAGCGGAGGAGGAGGCAGCGGAGGTGGAGGGTCGGATATCGAACTCACTCAGTCCCCAGCAATCATGTCCGCTTCACCGGGAGAAAAGGTGACCATGACTTGCTCGGCCTCCTCGTCCGTGTCATACATGCACTGGTACCAACAAAAATCGGGGACCTCCCCTAAGAGATGGATCTACGATACCAGCAAACTGGCTTCAGGCGTGCCGGGACGCTTCTCGGGTTCGGGGAGCGGAAATTCGTATTCGTTGACCATTTCGTCCGTGGAAGCCGAGGACGACGCAACTTATTACTGCCAACAGTGGTCAGGCTACCCGCTCACTTTCGGAGCCGGCACTAAG CTGGAGATC393 Ss1 (full)ATGGCCCTCCCTGTCACCGCCCTGCTGCTTCCGCTGGCTCTTCTGCTCCACGCCGCTCGGCCCCAAGTCCAGCTCCAGCAGTCGGGCCCAGAGTTGGAGAAGCCTGGGGCGAGCGTGAAGATCTCATGCAAAGCCTCAGGCTACTCCTTTACTGGATACACGATGAATTGGGTGAAACAGTCGCATGGAAAGTCACTGGAATGGATCGGTCTGATTACGCCCTACAACGGCGCCTCCAGCTACAACCAGAAGTTCAGGGGAAAGGCGACCCTTACTGTCGACAAGTCGTCAAGCACCGCCTACATGGACCTCCTGTCCCTGACCTCCGAAGATAGCGCGGTCTACTTTTGTGCACGCGGAGGTTACGATGGACGGGGATTCGACTACTGGGGCCAGGGAACCACTGTCACCGTGTCGAGCGGAGGCGGAGGGAGCGGAGGAGGAGGCAGCGGAGGTGGAGGGTCGGATATCGAACTCACTCAGTCCCCAGCAATCATGTCCGCTTCACCGGGAGAAAAGGTGACCATGACTTGCTCGGCCTCCTCGTCCGTGTCATACATGCACTGGTACCAACAAAAATCGGGGACCTCCCCTAAGAGATGGATCTACGATACCAGCAAACTGGCTTCAGGCGTGCCGGGACGCTTCTCGGGTTCGGGGAGCGGAAATTCGTATTCGTTGACCATTTCGTCCGTGGAAGCCGAGGACGACGCAACTTATTACTGCCAACAGTGGTCAGGCTACCCGCTCACTTTCGGAGCCGGCACTAAGCTGGAGATCACCACTACCCCAGCACCGAGGCCACCCACCCCGGCTCCTACCATCGCCTCCCAGCCTCTGTCCCTGCGTCCGGAGGCATGTAGACCCGCAGCTGGTGGGGCCGTGCATACCCGGGGTCTTGACTTCGCCTGCGATATCTACATTTGGGCCCCTCTGGCTGGTACTTGCGGGGTCCTGCTGCTTTCACTCGTGATCACTCTTTACTGTAAGCGCGGTCGGAAGAAGCTGCTGTACATCTTTAAGCAACCCTTCATGAGGCCTGTGCAGACTACTCAAGAGGAGGACGGCTGTTCATGCCGGTTCCCAGAGGAGGAGGAAGGCGGCTGCGAACTGCGCGTGAAATTCAGCCGCAGCGCAGATGCTCCAGCC

TABLE 4Amino acid sequences for the heavy chain (HC) CDR1, CDR2, and CDR3regions of human anti-mesothelin scFvs SEQ SEQ SEQ ID ID ID Descrip.HC-CDR1 NO: HC-CDR2 NO: HC-CDR3 NO: M1 GYTFTGYYMH 136 RINPNSGGTNYAQKFQG155 GRYYGMDV 175 M2 GYTFTGYYMH 136 WINPNSGGTNYAQKFQG 156DLRRTVVTPRAYYGMDV 176 M3 GYTFTGYYMH 136 WINPNSGGTNYAQKFQG 156GEWDGSYYYDY 177 M4 GFTFSSYWMH 137 RINTDGSTTTYADSVEG 157 GHWAV 178 M5GYTFTDYYMH 138 WINPNSGGTNYAQKFQG 156 GWDFDY 179 M6 GYTFTSYYMH 139IINPSGGSTSYAQKFQ 158 YRLIAVAGDYYYYGMDV 180 M7 GFTFSSYAMH 140VISYDGSNKYYADSVKG 274 WKVSSSSPAFDY 181 M8 GYPFTGYSLH 141WINPNSGGTNYAQKFQG 159 DHYGGNSLFY 182 M9 GYTFTSYYMH 142 IINPSGGSTGYAQKFQG160 GGYSSSSDAFDI 183 M10 GYTFTSYGIS 143 WISAYNGNTNYAQKLQ 161VAGGIYYYYGMDV 184 M11 GYTFTGYYMH 144 WINPNSGGTNYAQNFQG 162 GWDFDY 185M12 GYTFTGYYMH 144 RINPNSGGTNYAQKFQG 163 TTTSYAFDI 186 M13 GFIFSDYYMG145 YIGRSGSSMYYADSVKG 164 SPVVAATEDFQH 187 M14 GFTFRGYYIH 146IINPSGGSRAYAQKFQG 165 TASCGGDCYYLDY 188 M15 GFTFDDYAMH 147GISWNSGSIGYADSVK 166 DGSSSWSWGYFDY 189 M16 GFTFDDYAMH 147GISWNSGSTGYADSVKG 167 DSSSWYGGGSAFDI 190 M17 GFTFDDYAMH 147GISWNSGSTGYADSVKG 167 DSSSWYGGGSAFDI 191 M18 GFTFSSYWMH 148RINSDGSSTSYADSVKG 168 TGWVGSYYYYMDV 192 M19 GFTFSSYGMH 149VISYDGSNKYYADSVKG 169 GYSRYYYYGMDV 193 M20 GFTFSSYAMS 150AISGSGGSTYYADSVKG 170 REAAAGHDWYFDL 194 M21 GYTFTSYYMH 151IINPSGGSTSYAQKFQG 171 SPRVTTGYFDY 195 M22 GDTSTRHYIH 152VINPTTGPATGSPAYAQMLQG 172 SVVGRSAPYYFDY 196 M23 GYTFTNYYMH 153IINPSGGYTTYAQKFQG 173 IRSCGGDCYYFDN 197 M24 GFSLSTAGVHVG 154LISWADDKRYRPSLRS 174 QGFDGYEAN 198 Ss1 GYSFTGYTMN 394 LITPYNGASSYNQKFRG395 GGYDGRGFDY 396

TABLE 5Amino acid sequences for the light chain (LC) CDR1, CDR2, and CDR3regions of human anti-mesothelin scFvs SEQ SEQ SEQ ID ID ID DescriptionLC-CDR1 NO: LC-CDR2 NO: LC-CDR3 NO: M1 RASQSVSSNFA 199 DASNRAT 223HQRSNWLYT 247 M2 QASQDISNSLN 200 DASTLET 224 QQHDNLPLT 248 M3RASQSINTYLN 201 AASSLQS 225 QQSFSPLT 249 M4 RASQSISDRLA 202 KASSLES 226QQYGHLPMYT 250 M5 RASQSIRYYLS 203 TASILQN 227 LQTYTTPD 251 M6RASQGVGRWLA 204 AASTLQS 228 QQANSFPLT 252 M7 RASQSVYTKYLG 205 DASTRAT229 QHYGGSPLIT 253 M8 RASQDSGTWLA 206 DASTLED 230 QQYNSYPLT 254 M9RASQDISSALA 207 DASSLES 231 QQFSSYPLT 255 M10 KSSHSVLYNRNNKNYLA 208WASTRKS 232 QQTQTFPLT 256 M11 RASQSIRYYLS 209 TASILQN 233 LQTYTTPD 257M12 RASQSISTWLA 210 KASTLES 234 QQYNTYSPYT 258 M13 RASQSVTSNYLA 211GASTRAT 235 QQYGSAPVT 259 M14 RASENVNIWLA 212 KSSSLAS 236 QQYQSYPLT 260M15 QGDALRSYYAS 213 GKNNRPS 237 NSRDSSGYPV 261 M16 QGDSLRSYYAS 214GRSRRPS 238 NSRDNTANHYV 262 M17 QGDSLRSYYAS 215 GKNNRPS 239 NSRGSSGNHYV263 M18 RASQSVSSNYLA 216 DVSTRAT 240 QQRSNWPPWT 264 M19 RASQSVYTKYLG 217DASTRAT 241 QHYGGSPLIT 265 M20 RASQSISSYLN 218 AASSLQS 242 QQSYSIPLT 266M21 RASQSISSWLA 219 KASSLES 243 QQYSSYPLT 267 M22 RASQGISDYS 220 AASTLQS244 QQYYSYPLT 268 M23 RASENVNIWLA 221 KSSSLAS 245 QQYQSYPLT 269 M24RASRGISSALA 222 DASSLES 246 QQSYSTPWT 270 Ss1 SASSSVSYMH 397 DTSKLAS 398QQWSGYPLT 399

EXAMPLES

The invention is further described in detail by reference to thefollowing experimental examples. These examples are provided forpurposes of illustration only, and are not intended to be limitingunless otherwise specified. Thus, the invention should in no way beconstrued as being limited to the following examples, but rather, shouldbe construed to encompass any and all variations which become evident asa result of the teaching provided herein.

Example 1: Generating CAR Constructs

ScFv to be used in the final CAR construct were derived from thepannings of human scFV libraries.

The amino acid sequences of the human scFv fragments are provided abovein Table 2 and the nucleic acid sequences of the human scFv fragmentsare provided above in Table 3. Full CAR constructs were generated usingthe scFv fragments of Table 2 with additional sequences, SEQ ID NOs:1-2, 6-7, 9-10, 12-13, 17-18, 20-21, and 36-37, shown below, to generatefull CAR constructs.

leader (amino acid sequence) (SEQ ID NO: 1) MALPVTALLLPLALLLHAARPleader (nucleic acid sequence) (SEQ ID NO: 12)ATGGCCCTCCCTGTCACCGCCCTGCTGCTTCCGCTGGCTCTTCTGCTCCA CGCCGCTCGGCCCCD8 hinge (amino acid sequence) (SEQ ID NO: 2)TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDCD8 hinge (nucleic acid sequence) (SEQ ID NO: 13)ACCACTACCCCAGCACCGAGGCCACCCACCCCGGCTCCTACCATCGCCTCCCAGCCTCTGTCCCTGCGTCCGGAGGCATGTAGACCCGCAGCTGGTGGGGCCGTGCATACCCGGGGTCTTGACTTCGCCTGCGATCD8 transmembrane (amino acid sequence) (SEQ ID NO: 6)IYIWAPLAGTCGVLLLSLVITLYC CD8 transmembrane (nucleic acid sequence)(SEQ ID NO: 17) ATCTACATTTGGGCCCCTCTGGCTGGTACTTGCGGGGTCCTGCTGCTTTCACTCGTGATCACTCTTTACTGT 4-1BB Intracellular domain (amino acid sequence)(SEQ ID NO: 7) KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL4-1BB Intracellular domain (nucleic acid sequence) (SEQ ID NO: 18)AAGCGCGGTCGGAAGAAGCTGCTGTACATCTTTAAGCAACCCTTCATGAGGCCTGTGCAGACTACTCAAGAGGAGGACGGCTGTTCATGCCGGTTCCCAGAGGAGGAGGAAGGCGGCTGCGAACTG CD3 zeta domain (amino acid sequence)(SEQ ID NO: 9) RVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDT YDALHMQALPPRCD3 zeta (nucleic acid sequence) (SEQ ID NO: 20)CGCGTGAAATTCAGCCGCAGCGCAGATGCTCCAGCCTACAAGCAGGGGCAGAACCAGCTCTACAACGAACTCAATCTTGGTCGGAGAGAGGAGTACGACGTGCTGGACAAGCGGAGAGGACGGGACCCAGAAATGGGCGGGAAGCCGCGCAGAAAGAATCCCCAAGAGGGCCTGTACAACGAGCTCCAAAAGGATAAGATGGCAGAAGCCTATAGCGAGATTGGTATGAAAGGGGAACGCAGAAGAGGCAAAGGCCACGACGGACTGTACCAGGGACTCAGCACCGCCACCAAGGACACCTATGACGCTCTTCACATGCAGGCCCTGCCGCCTCGGCD3 zeta domain (amino acid sequence;NCBI Reference Sequence NM_000734.3) (SEQ ID NO: 10)RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDT YDALHMQALPPRCD3 zeta (nucleic acid sequence; NCBI Reference Sequence NM_000734.3);(SEQ ID NO: 21) AGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGC IgG4 Hinge (amino acid sequence)(SEQ ID NO: 36) ESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGKM IgG4 Hinge (nucleotide sequence)(SEQ ID NO: 37) GAGAGCAAGTACGGCCCTCCCTGCCCCCCTTGCCCTGCCCCCGAGTTCCTGGGCGGACCCAGCGTGTTCCTGTTCCCCCCCAAGCCCAAGGACACCCTGATGATCAGCCGGACCCCCGAGGTGACCTGTGTGGTGGTGGACGTGTCCCAGGAGGACCCCGAGGTCCAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCACAACGCCAAGACCAAGCCCCGGGAGGAGCAGTTCAATAGCACCTACCGGGTGGTGTCCGTGCTGACCGTGCTGCACCAGGACTGGCTGAACGGCAAGGAATACAAGTGTAAGGTGTCCAACAAGGGCCTGCCCAGCAGCATCGAGAAAACCATCAGCAAGGCCAAGGGCCAGCCTCGGGAGCCCCAGGTGTACACCCTGCCCCCTAGCCAAGAGGAGATGACCAAGAACCAGGTGTCCCTGACCTGCCTGGTGAAGGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAACGGCCAGCCCGAGAACAACTACAAGACCACCCCCCCTGTGCTGGACAGCGACGGCAGCTTCTTCCTGTACAGCCGGCTGACCGTGGACAAGAGCCGGTGGCAGGAGGGCAACGTCTTTAGCTGCTCCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGAGCCTGAGCCTGTCCCTGGGCAAGATGThese clones all contained a Q/K residue changein the signal domain of the co-stimulatory domainderived from CD3 zeta chain.

The CAR scFv fragments were then cloned into lentiviral vectors tocreate a full length CAR construct in a single coding frame, and usingthe EF1 alpha promoter for expression (SEQ ID NO: 11).

EF1 alpha promoterCGTGAGGCTCCGGTGCCCGTCAGTGGGCAGAGCGCACATCGCCCACAGTCCCCGAGAAGTTGGGGGGAGGGGTCGGCAATTGAACCGGTGCCTAGAGAAGGTGGCGCGGGGTAAACTGGGAAAGTGATGTCGTGTACTGGCTCCGCCTTTTTCCCGAGGGTGGGGGAGAACCGTATATAAGTGCAGTAGTCGCCGTGAACGTTCTTTTTCGCAACGGGTTTGCCGCCAGAACACAGGTAAGTGCCGTGTGTGGTTCCCGCGGGCCTGGCCTCTTTACGGGTTATGGCCCTTGCGTGCCTTGAATTACTTCCACCTGGCTGCAGTACGTGATTCTTGATCCCGAGCTTCGGGTTGGAAGTGGGTGGGAGAGTTCGAGGCCTTGCGCTTAAGGAGCCCCTTCGCCTCGTGCTTGAGTTGAGGCCTGGCCTGGGCGCTGGGGCCGCCGCGTGCGAATCTGGTGGCACCTTCGCGCCTGTCTCGCTGCTTTCGATAAGTCTCTAGCCATTTAAAATTTTTGATGACCTGCTGCGACGCTTTTTTTCTGGCAAGATAGTCTTGTAAATGCGGGCCAAGATCTGCACACTGGTATTTCGGTTTTTGGGGCCGCGGGCGGCGACGGGGCCCGTGCGTCCCAGCGCACATGTTCGGCGAGGCGGGGCCTGCGAGCGCGGCCACCGAGAATCGGACGGGGGTAGTCTCAAGCTGGCCGGCCTGCTCTGGTGCCTGGCCTCGCGCCGCCGTGTATCGCCCCGCCCTGGGCGGCAAGGCTGGCCCGGTCGGCACCAGTTGCGTGAGCGGAAAGATGGCCGCTTCCCGGCCCTGCTGCAGGGAGCTCAAAATGGAGGACGCGGCGCTCGGGAGAGCGGGCGGGTGAGTCACCCACACAAAGGAAAAGGGCCTTTCCGTCCTCAGCCGTCGCTTCATGTGACTCCACGGAGTACCGGGCGCCGTCCAGGCACCTCGATTAGTTCTCGAGCTTTTGGAGTACGTCGTCTTTAGGTTGGGGGGAGGGGTTTTATGCGATGGAGTTTCCCCACACTGAGTGGGTGGAGACTGAAGTTAGGCCAGCTTGGCACTTGATGTAATTCTCCTTGGAATTTGCCCTTTTTGAGTTTGGATCTTGGTTCATTCTCAAGCCTCAGACAGTGGTTCAAAGTTTTTTTCTTCCATTTCAGGTGTCGTGA (SEQ ID NO: 11). Gly/Ser (SEQ ID NO: 25)GGGGSGly/Ser (SEQ ID NO: 26): This sequence may encompass 1-6 “Gly Gly Gly GlySer” repeating units GGGGSGGGGS GGGGSGGGGS GGGGSGGGGSGly/Ser (SEQ ID NO: 27) GGGGSGGGGS GGGGSGGGGS Gly/Ser (SEQ ID NO: 28)GGGGSGGGGS GGGGS Gly/Ser (SEQ ID NO: 29) GGGS PolyA (SEQ ID NO: 30)This sequence may encompass 50-5000 adenines.aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 60aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 120aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 180aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 240aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 300aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 360aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 420aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 480aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 540aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 600aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 660aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 720aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 780aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 840aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 900aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 960aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 1020aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 1080aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 1140aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 1200aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 1260aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 1320aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 1380aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 1440aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 1500aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 1560aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 1620aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 1680aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 1740aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 1800aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 1860aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 1920aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 1980aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 2040aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 2100aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 2160aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 2220aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 2280aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 2340aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 2400aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 2460aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 2520aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 2580aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 2640aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 2700aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 2760aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 2820aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 2880aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 2940aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 3000aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 3060aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 3120aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 3180aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 3240aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 3300aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 3360aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 3420aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 3480aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 3540aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 3600aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 3660aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 3720aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 3780aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 3840aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 3900aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 3960aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 4020aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 4080aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 4140aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 4200aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 4260aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 4320aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 4380aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 4440aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 4500aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 4560aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 4620aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 4680aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 4740aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 4800aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 4860aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 4920aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 4980aaaaaaaaaa aaaaaaaaaa  5000 PolyA (SEQ ID NO: 31)tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt 60tttttttttt tttttttttt tttttttttt tttttttttt 100 PolyA (SEQ ID NO: 32)This sequence may encompass 50-5000 thymines.tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt 60tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt 120tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt 180tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt 240tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt 300tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt 360tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt 420tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt 480tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt 540tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt 600tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt 660tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt 720tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt 780tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt 840tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt 900tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt 960tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt 1020tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt 1080tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt 1140tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt 1200tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt 1260tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt 1320tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt 1380tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt 1440tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt 1500tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt 1560tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt 1620tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt 1680tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt 1740tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt 1800tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt 1860tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt 1920tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt 1980tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt 2040tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt 2100tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt 2160tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt 2220tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt 2280tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt 2340tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt 2400tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt 2460tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt 2520tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt 2580tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt 2640tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt 2700tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt 2760tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt 2820tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt 2880tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt 2940tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt 3000tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt 3060tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt 3120tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt 3180tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt 3240tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt 3300tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt 3360tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt 3420tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt 3480tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt 3540tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt 3600tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt 3660tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt 3720tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt 3780tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt 3840tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt 3900tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt 3960tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt 4020tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt 4080tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt 4140tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt 4200tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt 4260tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt 4320tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt 4380tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt 4440tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt 4500tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt 4560tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt 4620tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt 4680tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt 4740tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt 4800tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt 4860tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt 4920tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt 4980tttttttttt tttttttttt  5000 PolyA (SEQ ID NO: 33)This sequence may encompass 100-5000 adenines.aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 60aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 120aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 180aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 240aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 300aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 360aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 420aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 480aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 540aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 600aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 660aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 720aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 780aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 840aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 900aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 960aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 1020aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 1080aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 1140aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 1200aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 1260aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 1320aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 1380aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 1440aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 1500aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 1560aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 1620aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 1680aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 1740aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 1800aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 1860aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 1920aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 1980aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 2040aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 2100aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 2160aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 2220aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 2280aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 2340aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 2400aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 2460aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 2520aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 2580aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 2640aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 2700aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 2760aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 2820aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 2880aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 2940aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 3000aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 3060aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 3120aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 3180aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 3240aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 3300aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 3360aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 3420aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 3480aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 3540aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 3600aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 3660aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 3720aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 3780aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 3840aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 3900aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 3960aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 4020aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 4080aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 4140aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 4200aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 4260aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 4320aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 4380aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 4440aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 4500aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 4560aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 4620aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 4680aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 4740aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 4800aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 4860aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 4920aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 4980aaaaaaaaaa aaaaaaaaaa  5000 PolyA (SEQ ID NO: 34)aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 60aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 120aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 180aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 240aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 300aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 360aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa  400 PolyA (SEQ ID NO: 35)aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 60aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 120aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 180aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 240aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 300aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 360aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 420aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 480aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 540aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 600aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 660aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 720aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 780aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 840aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 900aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 960aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 1020aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 1080aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 1140aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 1200aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 1260aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 1320aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 1380aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 1440aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 1500aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 1560aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 1620aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 1680aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 1740aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 1800aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 1860aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 1920aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 1980aaaaaaaaaa aaaaaaaaaa  2000Gly/Ser (SEQ ID NO: 38): This sequence may encompass 1-10 “Gly Gly Gly Ser”repeating units GGGSGGGSGG GSGGGSGGGS GGGSGGGSGG GSGGGSGGGS

Example 2: Expression and Characterization of Human Anti-Mesothelin scFvConstructs for CAR Therapy

Additional analysis was performed to characterize the humananti-mesothelin scFv constructs, e.g., mass spectrometry analysis, sizeexclusion chromatography, and surface plasmon resonance (SPR). Bindingaffinity and epitope binding (compared to SS1 epitope) on theextracellular domain of mesothelin was determined by SPR. The assays andresults from this analysis are described below.

Expression of scFv Candidates and Biotinylated Human Mesothelin

To assess the binding and biophysical characteristics of humananti-mesothelin scFvs identified by phage panning, scFv constructs weretransiently produced and purified from HEK293F cells along with humanmesothelin extracellular domain (human MSLN ECD). SS1 scFv, which is amurine anti-mesothelin scFv, was also produced as a reference. The humananti-mesothelin scFvs tested were M5, M11, M12, M14, M16, M17, M21 andM23 (see Table 14). The plasmids encoding the amino acids for the scFvconstructs of M5 (SEQ ID NO: 43), M11 (SEQ ID NO: 49), M12 (SEQ ID NO:50), M14 (SEQ ID NO: 52), M16 (SEQ ID NO: 54), M17 (SEQ ID NO: 55), M21(SEQ ID NO: 59), M23 (SEQ ID NO: 61) and SS1 (SEQ ID NO: 275) and humanMSLN ECD (SEQ ID NO: 276) were synthesized externally. ScFvs wereproduced with a 7× (SEQ ID NO: 400) or 8×His (SEQ ID NO: 401) Tag on theC-terminus of the constructs. The human scFv constructs (M5, M11, M12,M14, M16, M17, M21, and M23) had a short linker sequence comprising thesequence GS linking the C-terminus of the scFv to the N-terminus of an8×His (SEQ ID NO: 401) Tag, e.g., GSHHHHHHHH (SEQ ID NO: 277). Humanmesothelin ECD was produced with a C-terminal Avi-Tag (SEQ ID NO: 276)and was site selectively biotinylated in vitro with BirA enzyme fromAvidity, LLC. Transient expression and purification in HEK293F cells wasperformed with standard methodology. For scFvs, briefly, 100 ml ofHEK293F cells at 3×106 cells/ml were transfected with 100 μg plasmid and300 μg polyethylenimine. The cells were incubated at 37° C. with 8% CO2and rotated at 80 rpm. After six days, the cells were harvested bycentrifugation at 3500 g for 20 minutes. The supernatant was purified bybinding the scFv to 200 μl Ni-NTA agarose beads (Qiagen) overnight at 4°C. The protein was eluted with 200 μl 300 mM imidazole, and dialyzedagainst phosphate buffered saline.

The sequence of the SS1 scFv with the His Tag is provided below:

(SEQ ID NO: 275) QVQLQQSGPELEKPGASVKISCKASGYSFTGYTMNWVKQSHGKSLEWIGLITPYNGASSYNQKFRGKATLTVDKSSSTAYMDLLSLTSEDSAVYFCARGGYDGRGFDYWGQGTTVTVSSGGGGSGGGGSGGGGSDIELTQSPAIMSASPGEKVTMTCSASSSVSYMHWYQQKSGTSPKRWIYDTSKLASGVPGRFSGSGSGNSYSLTISSVEAEDDATYYCQQWSGYPLTFGAGTKLEIEFGSHHHHHHH

The sequence of the human mesothelin ECD with the C-terminal Avi-Tag isprovided below:

(SEQ ID NO: 276) DAAQPAASEVEKTACPSGKKAREIDESLIFYKKWELEACVDAALLATQMDRVNAIPFTYEQLDVLKHKLDELYPQGYPESVIQHLGYLFLKMSPEDIRKWNVTSLETLKALLEVNKGHEMSPQVATLIDRFVKGRGQLDKDTLDTLTAFYPGYLCSLSPEELSSVPPSSIWAVRPQDLDTCDPRQLDVLYPKARLAFQNMNGSEYFVKIQSFLGGAPTEDLKALSQQNVSMDLATFMKLRTDAVLPLTVAEVQKLLGPHVEGLKAEERHRPVRDWILRQRQDDLDTLGLGLQGTRGSHHHHHHEFRHDSGLNDIFEAQKIEWHE

Mass Spectrometry Analysis

To confirm identity, purified scFvs were analyzed with high-performanceliquid chromatography coupled to mass spectrometry (HPLC-MS). 1 μg eachwas injected onto a Poros R1/10 2.1 mm×100 mm column (Life Technologies)heated to 60° C. Separation was performed on a Waters BioAcquity UPLC.Mobile Phase A was 0.1% formic acid; mobile phase B was 0.1% formic acidin 25% acetonitrile, 75% isopropanol. The scFvs were eluted at 0.5mL/min using a gradient from 25-50% mobile phase B in 15 minutes. Massspectrometry detection was performed with a Waters Xevo-Tof instrumentoperating in electrospray positive ion mode scanning from 600-4000 m/zwith a cone voltage ramp of 20-50V. The resulting mass spectra wereaveraged across the width of the peak and deconvoluted using the MaxEntlalgorithm from Waters to determine the masses of the expressed scFvs.

Size Exclusion Chromatography Analysis

Size exclusion chromatography was performed to determine theoligomerization states of the expressed scFvs. 25 μg each was injectedonto a TSKGel Super SW3000 4.6 mm×300 mm column (Tosoh Bioscience)heated to 35° C. The scFvs were eluted at 0.3 mL/min in 750 mM arginine,1 mM EDTA, 20 mM sodium phosphate, 250 mM sodium chloride, pH 7.2; theUV absorbance was monitored at 280 nm.

Surface Plasmon Resonance (SPR)

Binding affinity of purified scFvs was measured on a Biacore T200system. Briefly, recombinant human biotinylated mesothelin ECD wasimmobilized on a streptavidin (SA) sensor chip surface at a density of150 RU. Purified scFv was injected over the chip under constant flowrate at three-fold serial dilutions. Association and dissociation ratesof the protein complex were monitored for 270 seconds and 400 seconds,respectively. Double referencing was performed against a blankimmobilized flow cell and a buffer blank. Affinity was determined with aLangmuir 1:1 binding model when possible, for those scFVs where accuratefitting was not possible due to fast on and off rates, the steady statemodel was used.

To determine the relative binding epitope of each of the scFvs incomparison to SS1, 50 nM SS1 was captured by biotinylated mesothelin ECDimmobilized on a streptavidin sensor chip at a contact time of 180seconds resulting in a relative density of 70 RU. Purified scFv at 100nM (M5, M11, M12, M14, M16, M17, M21, or M23) was then immediatelyinjected over the chip to minimize dissociation of the SS1/mesothelincomplex. Binding of the secondary scFv was monitored for 270 seconds.

Results

The observed masses for the scFvs, determined by HPLC-MS, wereconsistent with the theoretical values based on the amino acidsequences. Expressed scFvs ranged from 43% monomer to >98% monomer basedupon analytical size exclusion chromatography. The selected scFvsrepresented a broad range of affinities from 0.9 nM to 114 nM comparedto SS1 control scFV which had an apparent affinity of 0.1 nM formesothelin ECD (Table 14). Representative SPR sensograms for SS1, M5,and M11 are shown in FIGS. 41A, B and C, respectively.

Relative epitope binning (FIG. 42) suggests M12, M14, M16, M17, M21, M23are competitive binders with SS1 to human mesothelin while M5 and M11appear to bind to a unique epitope, as judged by the increased responsein the assay upon their injection.

TABLE 14 Characteristics of anti-human Mesothelin scFvs Identity SEC¹Affinity² Theor. Obs. % ka kd KD Sample Mass Mass Monomer Fit (1/Ms)(1/s) (nM) M5 26866 26865  82% 1:1 Binding 4.20E+04 1.13E−03 26.9 M11³26949 26948 ND³ Steady State Affinity — — 64.7 M12 27261 27260  70%Steady State Affinity — — 307 M14 27420 27422 74.5% 1:1 Binding 2.75E+062.51E−03 0.9 M16 27127 27128 94.5% 1:1 Binding 1.74E+05 3.95E−03 22.7M17 26890 26891 >98% 1:1 Binding 5.22E+05 1.45E−02 27.8 M21 2741227411 >98% Steady State Affinity — — 110 M23 27602 27604  91% SteadyState Affinity — — 114 SS1 26504 26503  43% 1:1 Binding 5.55E+065.60E−04 0.1 ¹High aggregate content/low % monomer may result in lessaccurate affinity determinations due to potential avidity effects.²Affinity was determined with 1:1 binding model when possible, for thosescFvs where accurate fitting was not possible due to fast on and offrates, steady state model was used. ³Not determined, low concentrationprecluded accurate determination of % monomer.

Example 3: Analysis and In Vitro Activity of Human scFv Bearing CARTs

Single chain variable fragments for anti-MSLN antibodies were clonedinto lentiviral CAR expression vectors with the CD3zeta chain and the4-1BB costimulatory molecule and the optimal constructs are selectedbased on the quantity and quality of the effector T cell responses ofMSLN CAR transduced T cells (“CART-MSLN” or “CART-MSLN T cells”) inresponse to MSLN expressing (“MSLN+”) targets. Effector T cell responsesinclude, but are not limited to, cellular expansion, proliferation,doubling, cytokine production and target cell killing or cytolyticactivity (degranulation).

Generation of CART-MSLN

The human scFv encoding lentiviral transfer vectors are used to producethe genomic material packaged into the VSVg psuedotyped lentiviralparticles. Lentiviral transfer vector DNA is mixed with the threepackaging components of VSVg, gag/pol and rev in combination withlipofectamine reagent to transfect them together in to Lenti-X 293Tcells (Clontech).

After 30 hours, the media was collected, filtered and stored at −80C.The therapeutic CART-MSLN were generated by starting with the blood froma normal apheresed donor whose naïve T cells are obtained by negativeselection for T cells, CD4+ and CD8+ lymphocytes. These cells areactivated by CD3×28 beads (Dynabeads® Human T-Expander CD3/CD28,Invitrogen) at a ratio of 1:3 in RPMI1640, 10% heat-inactivated fetalcalf serum (FCS), 2 mM L-glutamine, 1× Penicillin/Streptomycin, 100 μMnon-essential amino acids, 1 mM NaPyruvate, 10 mM Hepes, and 55 μM2-mercaptoethanol) at 37° C., 5% CO₂. T cells were cultured at 1×10⁶ Tcells in 0.5 mL medium per well of a 24-well plate. After 24 hours, theT cells are blasting and 0.5 mL of non-concentrated, or smaller volumesof concentrated viral supernatant is added. The T cells begin to dividein a logarithmic growth pattern, which is monitored by measuring thecell counts per mL, and T cells are diluted in fresh medium every twodays. As the T cells begin to rest down after approximately 10 days, thelogarithmic growth wanes. The combination of slowing growth rate and Tcell size approaching ˜300 fl determines the state for T cells to becryopreserved for later analysis.

Before cryopreserving, percentage of cells transduced (expressing themesothelin-specific CAR on the cell surface) and their relativefluorescence intensity of that expression were determined by flowcytometric analysis on a FACS-CantoII or FACS Fortessa. Comparinghistogram plots of relative fluorescent intensity from that FACS showedthe percentage of transduced T cells. The virus transduction showcomparable expression levels correlating with transduction efficiency,percent cells transduced. The results indicate that there is nodetectable negative effect of the human scFv bearing CAR-MSLN on thecells ability to expand normally when compared to the untransduced Tcells (“UTD”) and SS1 CART-MSLN.

Evaluating Cytolytic Activity and Cytokine Secretion of CART-MSLNRedirected T Cells.

To evaluate the functional abilities of CART-MSLN T cells to kill andsecrete cytokines, the cells were thawed and allowed to recoverovernight. In addition to the human scFv bearing CART-MSLN, theCART-MSLN bearing the murine scFv “SS1” (see WO2013/040577) was used asa control. The control SS1 scFV bearing CART-MSLN (also referred to asSS1 CART-MSLN) was used in all assays to compare assay variation and/oract as a control. The SS1 scFV bearing CART cells, as well as all theother human scFv bearing MSLN-CARTs, were produced in research grade(i.e., not clinical grade) manufacturing conditions. CD19-BBz orIsol-BBz was used as non-targeting CAR for background CART effect.

T cell killing was directed towards K562, a chronic myelogenous leukemiacell line. K652 MSLN+(K562-Meso) cells were generated by transduction.Non-MSLN expressing K5652 were used as a control. For the flow basedcytotoxicity assay, the target cells are stained with Carboxyfluoresceinsuccinimidyl ester (CSFE) quantitate their presence. The cytolyticactivities of CART-MSLN were measured at a titration of effector:targetcell ratios of 10:1, 3:1, and 1:1 where effectors were defined as Tcells expressing the anti-MSLN chimeric receptor. Assays were initiatedby mixing an appropriate number of T cells with a constant number oftargets cells. After 20 hours, the plates were centrifuged and the totalvolume of each mixture was removed. The remaining cell pellet in eachwell was washed and cells were stained with live/dead marker 7AAD. Afterthe final wash, the pelleted cells were re-suspended in a specificvolume with a predetermined number of counting beads. Cell staining datawas collected by Cantoll flow cytometry and analyzed with FloJo softwareusing beads to quantitate results.

Plots from 20 hour flow-based killing assays using titrating Effector toTarget (E:T) ratios with effector CART-MSLN cells targeting CSFE labeledK562 (FIG. 3A. non-expressing MSLN controls) and K562-Meso (FIG. 3B,K562 cells transduced to express MSLN). Comparing these killing curves,titrating the amount of effector cells shows that those cells expressingMSLN are destroyed. T cells from the same donor and that were transducedwith either human scFv bearing CAR-MSLN cells or SS1 CAR-MSLN cells wereable to kill selectively MSLN+ targets. The cytolytic activities of allCART-MSLN cells are similar and comparable to the murine SS1 CART-MSLN.They show that all CART-MSLN cells become Mesothelin reactive upontransduction and gain the ability to kill target cells expressing highlevels of Mesothelin. For measuring cytokine production of human scFvbearing CART-MSLN, cells were thawed and allowed to recover overnight.In addition to the humanized CART-MSLN, the SS1 CART-MSLN (murine scFv)was used for comparative purposes. CD19-BBz CART was used as anon-targeting control for background CART cell effect. SS1 CART-MSLN wasused in all assays to compare assay variation. The T cells were directedtowards K562, a chronic myelogenous leukemia cell line (both MSLN+ andMSLN), the MLSN-expressing ovarian cancer cell line Ovcar8, and thepancreatic cancer lines SW1990 and Panc0203. When analyzing MSLNexpression by flow cytometry, we detected 10-fold lower expression ofMSLN on Ovcar8 compared to MSLN+K562 cells; SW1990 expressed 10-foldless than Ovcar8. MSLN expression on Panc0203 was not detectable by flowcytometry, but was positive by RNA analysis. In addition, PMA/Ionomycinwas used to evaluate the potential of T cells to respond to theendogenous immunological signals. The assay tests only aneffector:target ratio of 1:1. The assay is run for 24 hours after mixingof the cells, when the media is removed for cytokine analysis using theIFN-γ CBA-Flex kit for human cytokine detection.

Background levels of cytokine produced from CART-MSLN cells afterexposure to the control K562 cells not expressing MSLN were analyzed.The potential cytokine secretion from stimulation of the T cells withPMA/Ionomycin indicates that the cell populations have slightly variablepotential to secrete cytokine. Differences were dealt with bynormalizing the specific IFN release over the maximum release(PMA/Ionomycin).

Data shows that most human scFv bearing CART-MSLN and the murine SS1CART-MSLN produced IFNγ when cultured with K562 overexpressig MSLN. Whencultuered with cancer cells endogenously expressing MSLN (at lowerlevels), only few CARTs responded (M5, 11, 14, 15, 16, 17 and SS1),while the two CARTs M5 and M11 showed superior IFNγ levels. The lowerendogenous MSLN levels they might better reflect naturally expressedlevels of mesothelin on tumor cells, those CART having those scFVs mighthave an enhanced therapeutic response than CARTs having the SS1construct.

Example 4: Clinical Trial for Lentiviral Generated CARTs

Patients will be selected from those have mesothelin expressing cancers,such as serious epithelial ovarian cancer, malignant pluralmesothelioma, or pancreatic cancer.

The manufacture and release testing of CART-MSLN cells will be performedby the Clinical Cell and Vaccine Production (CVPF) at the University ofPennsylvania. CART-MSLN product will be manufactured from the patients'apheresis product.

An about 10-liter apheresis procedure will be carried out at theUniversity of Pennsylvania Apheresis center (4 weeks prior to first doseof CAR T cells). PBMCs from a patient are obtained for CAR T cellsduring this procedure. From a single leukapheresis, the intention is toharvest at least 5×10⁹ white blood cells to manufacture CAR T cells.Baseline blood leukocytes for FDA look-back requirements and forresearch are also obtained and cryopreserved. The mesothelin-modified Tcell product is expected to be ready for release approximately 4 weekslater.

Lentiviral generated CARTs were made as described in Example 2 usingcells isolated from each patient, i.e., autologous T cells. TheCART-MSLN made from each patients T cells express human scFvs (includingM5 and M11), or SS1.

At the end of cell cultures, the cells are cryopreserved in infusiblecryomedia in bags. The dose is 1-5×10⁷ CAR T cells for Cohort 1, 1-5×10⁸CAR transduced cells for Cohort 2 and 3, calculated as a range of 2-50%transduced cells in total cells. Bags containing autologous modified CART cell products will be stored in a controlled and monitored freezer atthe CVPF at the Hospital of the University of Pennsylvania. Infusionbags will be stored in the freezer until needed.

Each dose will be packed and cryopreserved in 1 infusion bag at in avolume dependent on the total cell number, a function of thetransduction efficiency; the minimum volume will be 10 mL per bag. Eachbag will contain an aliquot (volume dependent upon total cell dose) ofcryomedia.

Cohort 1 subjects (N=3-6) will receive a single flat intravenous dose ofautologous 1-5×10⁷ CART-MSLN cells on day 0. Cohort 2 subjects (N=3-6)will receive a single flat intravenous dose of autologous 1-5×10⁸CART-MSLN cells on day 0. Subjects in Cohort 1 and 2 will not receiveany lymphodepleting chemotherapy prior to CART-MSLN cells. Cohort 3(N=6) will receive 1.5 grams/m² of cyclophosphamide intravenously twodays prior to a single flat intravenous dose of autologous 1-5×10⁸CART-MSLN cells.

It is anticipated that patients receiving cyclophosphamide mayexperience nausea and vomiting as a side effect of the treatment.Anti-emetic prophylaxis premedication for nausea can be administeredprior to infusion of chemotherapy according to the institutionalstandards. Choice of specific agent will be left to the discretion ofthe investigator, though corticosteroids should not be used due to theirimmunosuppressive effects.

Side effects following T cell infusions may include transient fever,chills, rigors, myalgias/arthralgias, headache, fatigue, and/or nausea.Subjects will be pre-medicated with acetaminophen 650 mg by mouth anddiphenhydramine hydrochloride by mouth or IV prior to CAR T cellinfusion. If Benadryl is contraindicated, an H2-blocker, such asranitidine, will be administered. These medications may be repeatedevery six hours as needed. A course of non-steroidal anti-inflammatorymedication may be prescribed if the patient continues to have fever notrelieved by acetaminophen. It is recommended that patients do notreceive systemic corticosteroids such as hydrocortisone, prednisone,prednisolone (Solu-Medrol), or dexamethasone (Decadron). Ifcorticosteroids are required for an acute infusional reaction, aninitial dose of hydrocortisone 100 mg is recommended.

-   -   One bag of CART-MSLN cells will be transported by the study        coordinator or CVPF stuff on dry ice from CVPF to the subject's        bedside in the CTRC.    -   Transfected T cells will be thawed by a member of CVPF staff in        a 37° C. water bath at subject's bedside. The bag will be gently        massaged until the cells have just thawed. If the CAR T cell        product appears to have a damaged or leaking bag, or otherwise        appears to be compromised, it should not be infused, and should        be returned to the CVPF as specified below.    -   Cells will be infused to the subject while cold within        approximately 10-15 minutes after thaw. The transfected T cells        will be infused intravenously rapidly through an 18 gauge latex        free Y-type blood set with 3-way stopcock. No leukocyte filter        at infusion. Dosing will take place by gravity infusion. If the        infusion rate by gravity is too slow, the transfected T cell        drug product may be drawn into a syringe via the stopcock and        manually infused at the required rate. There should be no frozen        clumps left in the bag.    -   Prior to the infusion, two individuals will independently verify        the information on the label of each bag in the presence of the        subject and confirm that the information correctly matches the        participant.    -   Patients will be monitored during and after infusion of the        transfected T cells. Temperature, blood pressure, heart rate,        respiratory rate and pulse oximetry will be obtained and        recorded immediately prior to dosing and every 15 minutes for 2        hours following infusion completion.    -   Emergency medical equipment (i.e. a crash cart) must be        available for an emergency situation during the infusion in the        event that the subject has an allergic response, or severe        hypotensive crisis, or any other reaction to the infusion.    -   If no symptoms occur and subject's vital signs remain normal 2        hours after the infusion, the subject will be discharged home        with instructions to return to the hospital should any symptoms        develop. If a vital sign measurement is not stable, it will        continue to be obtained approximately every 15 minutes until the        subject's vital signs stabilize or the physician releases the        patient. The subject will be asked not to leave until the        physician considers that it is safe for him or her to do so.    -   Within 60 minutes (±5 minutes) following completion of        transduced CAR T cell dosing, a blood sample will be obtained        for a baseline determination of transduced CAR T cell number and        cytokine levels.    -   Subjects will be instructed to return to the CTRC or Perelman        Center in 24 hours after the first infusion for blood tests and        follow-up examination according to the SOE.

For CARTs bearing scFv doses of cells can be repeated as needed.

Descriptive statistics will be applied to determine the relativepersistence and trafficking to blood (and optionally tumor) of theCART-MSLN cells. Data regarding the number of CAR T cells in blood, HAMAlevels, and the tracking of soluble biomarker levels will be presentedgraphically. Correlations with radiographic and other standard measuresof tumor burden will be determined. We will compute 95% confidenceintervals for proportions and means.

Adverse events will be collected and evaluated for all patients duringthe protocol specified AE reporting periods. AEs will be graded forseverity using the National Cancer Institute (NCI)—Common ToxicityCriteria (v4). All adverse events will be described and exact 95%confidence intervals will be produced for adverse event rates, bothoverall and within major categories. The data will be monitoredcontinuously for evidence of excessive toxicity. Results will betabulated and summarized.

Rates of clinical responses will be summarized in exact 95% confidenceintervals. Distributions of progression-free and overall survival andduration of clinical response will be presented graphically usingKaplan-Meier curves. The two-year survival rates will be presented.Preliminary evidence of efficacy will be determined by monitoring tumorresponse rates in those subjects with measurable disease. Tumor responsewill be assessed using radiographic imaging (i.e. CT imaging) and serumbiomarkers at Day 28, Months 3 and 6 after infusion.

-   -   Radiographic responses will be measured according to Response        Evaluation Criteria in Solid Tumors (RECIST 1.1), modified        RECIST criteria for pleural mesothelioma, and Immune-Related        Response Criteria (iRRC).    -   Serum biomarker responses will be evaluated according to        standards for each disease; additionally all subjects will have        SMRP measured. For example, subjects with pancreatic cancer will        have monitoring in levels of CA19-9, CEA and serum mesothelin        related protein [SMRP]) following CAR T cell administration.        Subjects with ovarian cancer will have serum levels of CA125        monitored, an also SMRP. Subjects with mesothelioma will have        serum levels of SMRP measured. In all cases, serum biomarkers        will not be used as the sole measurement of tumor response,        given that CART-MSLN cells may affect the assays used in these        biomarker measurements.    -   Data will be analyzed descriptively for overall response rates,        progression-free survival, and overall survival.

PFS and OS up to 2 years post-infusion or until subjects initiate acancer-related therapy will be evaluated.

Safety assessments including blood monitoring of cytokine levels will beperformed. Subjects will be enrolled serially. Infusions will bestaggered to allow 28 days of safety assessment between enrolledsubjects in each cohort.

Example 5: Clinical Trial for RNA Generated CARTs

Autologous T cells are engineered to express an extracellular a SS1 CARconstruct that recognized mesothelin, along with transmembrane domain,in addition to human 4-1BB and CD3zeta signaling domains describedabove. See, e.g., WO2013/040577, Example 1.

Production of CAR-Containing Nucleic Acids

SS1-CARs are constructed according as follows. CARs containing humananti-mesothelin scFvs are also constructed using similar methods.

Two different plasmids were utilized to clone the ssl.bbz fragment. Themesothelin scFv fragment (ssl) was first cloned by the TranslationalResearch Program (TRP) laboratory from the previously publishedconstruct of Dr. Pastan (Chowdhury et al., 1998). The human CD8α hingeand transmembrane domain together with 41BB and CD3ζ sequence was clonedby PCR from the pELNS.CD19-BB-ζ plasmid described previously (Milone etal., 2009). Sequences for the hinge, transmembrane, and intracellularsignaling domains are disclosed herein. The ssl.bbz fragment was firstcloned in pGEM.GFP.64A vector (provided by Eli Gilboa and described inBoczkowsk, D et al., 2000). This vector was modified by addition of two3′UTR beta globin repeats and 150 bp of polyA sequence (SEQ ID NO:271)(replacing the 64 polyA sequence (SEQ ID NO: 273) in pGEM.GFP.64A)for enhanced transgene expression (Holtkamp 2006). The GMP-compliantplasmid for clinical use was derived by subcloning thessl.bbz.2bgUTR.150A fragment from pGEM into the pDrive vector. ThepDrive cloning vector (Qiagen) is designed for highly efficient cloningof PCR products through UA hybridization. It allows for both ampicillinand kanamycin selection of recombinant clones, and comes with universalsequencing primer sites, and both T7 and SP6 promoters for in vitrotranscription. First, ssl.bbz.2bgUTR.150A was cut from pGEM vector byHind III and NdeI (Fill-in blunt) and subcloned into pDrive cut by KpnIand NotI (Fill-in blunt). The insert with correct orientation wassequence confirmed to generate pDrive.ssl.bbz.2bgUTR.150A. Ampicillinresistance gene in pDrive vectors was deleted by double digestion withAhdI and BciVI. To eliminate potential aberrant proteins translated frominternal open reading frames (ORF) inside the CAR ORFs, all internal ORFthat were larger than 60 bp in size were mutated by mutagenesis PCR,while the ORF of ss1 CAR was maintained intact. The resulting plasmidwas designated pD-A.ss1.bbz.OF.2bg.150A, as shown in FIG. 1.

Production of CAR nucleic acids for introduction into autologous T cellswere prepared as follows. The final pD-A.ss1.bbz.OF.2bg.150A constructwas introduced into OneShot TOP10 Chemically Competent E Coli cells(Invitrogen) as per CVPF SOP 1188. Up to 10 mg plasmid DNA prepared asone batch was generated using the QlAfilter Plasmid Giga DNA isolationkit as per SOP 1191, from two 1.25 liters of LB-media containing 100μg/ml kanamycin. 1 mg of DNA at a time was linearized with Spelrestriction enzyme overnight at 37° C. Linearization was confirmed bygel electrophoresis prior to large scale purification using the QiagenPlasmid Maxi Kit.

RNA was transcribed from the plasmids by utilizing the mScript mRNAsystem and was isolated using the RNeasy Maxi kit (Qiagen). The in vitrotranscribed RNA was cryopreserved in aliquots of 0.5 mL at aconcentration of 1 mg/mL. RNA quality and quantity was analyzed by 1%agarose gel electrophoresis after 15 min denaturation at 70° C. in mRNAdenaturation buffer (Invitrogen, Carlsbad, Calif.) and quantified by UVspectrophotometry (0D260/280).

CAR T Cells Product Manufacturing.

A 7 to 12-liter apheresis procedure is performed about 4 weeks prior tofirst dose of CAR T cells. PBMC are obtained for CAR T cells during thisprocedure. From a single leukapheresis, the intention is to harvest atleast 5×109 white blood cells to manufacture CAR T cells.

CD3+ T-cells are enriched from a leukapheresis product by depletion ofmonocytes via counterflow centrifugal elutriation on the CaridianBCTElutra, which employs a single use closed system disposable set. On day0, the T cell manufacturing process is initiated with activation withanti-CD3/CD28 monoclonal antibody-coated magnetic beads, and expansionis initiated in a static tissue culture bag. At day 5, cells will betransferred to a WAVE bioreactor if needed for additional expansion. Atthe end of the culture, cells are depleted of the magnetic beads,washed, and concentrated using the Haemonetics Cell Saver system. Thepost-harvest cells are incubated overnight at 37° C. for electroporationthe next morning. Cells are washed and resuspended in ElectroporationBuffer (Maxcyte) and loaded into the Maxcyte processing assembly. Cellsare electroporated with the ss1 RNA, and allowed to recover for 4 hoursand then formulated in infusible cryopreservation media.

Packaging

1×10⁸ or 1×10⁹±20% modified CAR T cells will be supplied in IV infusionbags maintained on wet ice. Each bag will contain an aliquot (volumedependent upon dose) of cryomedia containing the following infusiblegrade reagents (% v/v): 31.25 plasmalyte-A, 31.25 dextrose (5%), 0.45NaCl, up to 7.50 DMSO, 1.00 dextran 40, and 5.00 human serum albumin.Bags can be frozen at −135° C.

CAR T Cells Product Stability

The ss1 CAR T cells will be cryopreserved 4 hours post-electroporation,and thawed and administered within a three month window after T cellmanufacturing. We have demonstrated that mesothelin scFv expression ofthe cryopreserved ss1CAR T cells approximately 30 days at ≤−130° C. was97.4%, almost identical to time of cryopreservation (96.9%), and othercryopreserved T cell products are stable for at least 6 months.Viability post-thaw, based on Trypan blue counts was 75.2% as comparedto 98.7%. The expression data suggests that the final product is stableduring storage for the trial, and that the sentinel vial for additionaldoses should meet release criteria of 70% viability and ≥20% CARexpression. Additional vials of ss1 CAR T cells will be thawed at 3, 6,9, and 12 months post cryopreservation, and viability and transgeneexpression tested to generate further product stability data.

Clinical trial procedure and results: A Phase I clinical trial ofautologous mesothelin re-directed T cells administered to patients withmalignant pleural mesothelioma is performed as follows. Similarprocedures can be utilized for a study for treating patients withpancreatic cancer.

Dosages

Cohort 1 patients (n=3) will receive a single infusion of 1×10⁸ usingflat dosing with anti-meso RNA CAR T cells on day 0 and one infusion of1×10⁹ RNA CAR T cells on day 7, providing the patients meet theprotocol-specified safety assessments before the day 7 infusion.

Cohort 2 patients (n=6) will be given 2 cycles of modified CAR T cells.One cycle consists of 3 infusions every other day (Monday, Wednesday,Friday). Cycle 1 consists of 3 doses of 1×10⁸ CAR T cells dosed on MWF(day 0, 2, 4); cycle 2 consists of 3 doses of 1×10⁹ CAR T cells dosed onMWF (day 7, 9, 11).

The target dose per infusion is 1×108 cells (one dose for Cohort 1 andweek 1 doses for Cohort 2) and 1×10⁹±20% cells (one dose for Cohort 1and week 2 doses for Cohort 2). The minimally acceptable dose is 1×10⁸.If the total cell expansion is lower than the total acceptable celldose, the patient may undergo a second apheresis in an attempt to expandmore cells and fulfill the total target dose. If there arecontraindications for the second apheresis or if the second apheresisand expansion fails to produce the minimally acceptable dose, the dosewill be deemed a manufacturing failure. For Cohort 1 patients, as manymesothelin CAR electroporated T cells as possible will bemanufactured/frozen to reduce the probability subjects will undergo asecond leukapheresis in the case they will follow Extended Cohort 1regimen.

Dose de-escalation will occur if more than one patient develops a DLTfrom modified CAR T cell infusion. In the event of DLT, we will dosede-escalate by 10-fold. Thus, if toxicity occurs during cycle 1 at 10⁸CAR T cells, then all infusions (doses 1 to 6) would be reduced to 10⁷CAR T cells. In the event of unmanageable toxicity at 10⁷ CAR T cells,the trial would be stopped.

A potential issue that can arise in patients being treated usingtransiently expressing CAR T cells (particularly with murine scFvbearing CARTs) is anaphylaxis after multiple treatments.

Without being bound by this theory, it is believed that such ananaphylactic response might be caused by a patient developing humoralanti-CAR response, i.e., anti-CAR antibodies having an anti-IgE isotype.It is thought that a patient's antibody producing cells undergo a classswitch from IgG isotype (that does not cause anaphylaxis) to IgE isotypewhen there is a ten to fourteen day break in exposure to antigen.Therefore, for transient CARTs (such as those generated by RNAelectroporation), CART infusion breaks should last more than ten tofourteen days.

In addition, using CARs with human (instead of murine) scFvs couldreduce the likelihood and intensity of a patient having an anti-CARresponse.

Study Procedure

The study consists of 1) a screening phase, 2) an intervention phaseconsisting of apheresis, CAR T cell infusion, side effect assessment,and optional tumor biopsy, and 3) follow up visits.

Screening and Baseline Assessment

An investigator must explain the nature of the study protocol and risksassociated with the protocol in detail to the subject. The subject mustsign and date the written informed consent prior to study participation.Informed consent process and date will be recorded in the subject'smedical record and on the CRF. Informed consent must be obtained beforeprotocol procedures are performed. If a procedure required for screeningwas performed prior to signing the informed consent and the proceduremeets the time limits of the protocol, this procedure may be used forthe screening evaluation.

The screening procedures (not required for determination of eligibility)will be completed within 6 weeks of first dose of CART cells (unlessotherwise noted in the SOE). In the event that a second apheresis isneeded to expand additional T cells and reach the target cell dose, thistime window will not cover all the procedures. Thus, in this specificsituation, a 6-8 week period is allowed for performing the baselinescans.

Screening procedures include:

-   -   Informed consent    -   Confirm an ECOG performance status ≤1    -   Determination of manufacturing efficiency (for the first 2        patients): a blood sample is sent to the CVPF to determine T        cell manufacturing feasibility. In approximately 1 week, the        CVPF will return a result as to whether the subjects PBMC are        likely to be adequate for large scale CAR T cell manufacturing        process. Results must be known before performing any additional        study procedures.    -   Complete medical history    -   Physical examination including vital signs, height, weight and        oxygen saturation    -   Review of concomitant medications    -   Hematology (WBC with differential, RBC, Hct, Hgb and platelets)    -   PT and PTT    -   Chemistry (sodium, potassium, chloride, bicarbonate, urea        nitrogen, creatinine, glucose, total protein, albumin, calcium,        alkaline phosphatase, total bilirubin, ALT, AST)    -   Viral screens: HIV, HCV, HBV and serology for CMV    -   Autoantibody panel: ANA, ESR    -   Urinalysis    -   12 lead Electrocardiogram (ECG)    -   Serum βHCG pregnancy (women of childbearing potential)    -   Spirometry and DLCO        -   Tumor assessment Chest CT Scan with contrast, other            radiographic evaluations as clinically indicated. PET/CT            scan is optional.        -   Pathological confirmation of epithelial or mixed (biphasic)            mesothelioma and stain for mesothelin by protocol specific            pathologist.

Leukapheresis

A 7 to 12-liter apheresis procedure will be carried out at theUniversity of Pennsylvania Apheresis center (4 weeks prior to first doseof CAR T cells). PBMC are obtained for CAR T cells during thisprocedure. From a single leukapheresis, the intention is to harvest atleast 5×109 white blood cells to manufacture CAR T cells. If the harvestis unsuccessful, patients will have the option to undergo a secondleukapheresis. Baseline blood leukocytes for FDA look-back requirementsand for research are also obtained and cryopreserved. Themesothelin-modified T cell product is expected to be ready for releaseapproximately 4 weeks later. All patients will have the standardleukapheresis screening prior to the procedure.

Pre-Infusion Assessment (Study Day −3, Prior to First Dose)

This safety assessment includes physical exam (e.g., vital signs,weight, etc.), review of concomitant medications, performace status,chemistry, CBC with differential, pregnancy test (urin) urinalysis, EKG,CXR, and research blood specimens.

CAR T Cell Administration

Prior to each CAR T cell infusion patients will have limited problemoriented PE, review of adverse events, ECOG performance status, CBC,chemistry, and urinalysis. Subjects will return to HUP 1 day after CAR Tcell infusion (D1, D3, D8 and D10) to have limited problem oriented PE,review of adverse events, concomitant medications, ECOG performancestatus, CBC and chemistry. On day 7, Cohort 1 patients will also have anEKG. On Day 14 Cohort 2 patients will also have a CXR and EKG.

CAR T cells will be administered as described above. Subjects' vitalsigns will be assessed and pulse oximetry will be done prior to dosing,at the end of the infusion and every 15 minutes thereafter for 2 hoursand until these are stable.

Post-Infusion Assessment

Cytokine levels in blood will be monitored at 1 h, 4 h, 1 day and 3 daysfollowing first infusion and at 1 h, 4 h, 1 day and 3 days, 7 days and14 days following the second infusion for patients of Cohort 1. Bloodcollection for engraftment analysis (flow cytometry and RT-PCR) will becollected for 21 days following the first infusion.

For Cohort 2 (and Extended Cohort 1) patients, cytokine levels will bemonitored at 1 h following each infusion and at additional time pointsaccording to the SOE. The primary safety and engraftment data iscollected for 35 days following the first (Day 0) CAR T celladministration.

1 day after the CAR T cell infusion, blood is drawn for flow cytometryand RT-PCR in order to evaluate for circulating CAR T cells. Peripheralblood samples will be processed to isolate: a) peripheral bloodmononuclear cells (PBMC) to evaluate immune cell phenotype and function,b) RNA to quantify short term persistence and distribution of infusedCAR T cells and c) serum to quantify and evaluate immune cell subset andsoluble immune and growth factors, evaluate the development ofanti-infused cell immune responses, and perform protoarray analyses toevaluate the development of humoral anti-tumor immune responses viaimmune epitope spreading.

Subjects will be assessed on month 2 and 3 (Cohort 1), and months 2, 3,and 6 following the last CAR T cell infusion (Cohort 2 and extendedCohort 1) for safety assessment as described above.

Tumor Biopsy and Mini-Apheresis

For Cohort 2 and extended Cohort 1: If tumor is visibly accessible oraccessible by image-guided biopsy, eligible subjects will undergo directtumor biopsy or biopsy under image guidance to evaluate the presence ofCAR T cells and mesothelin expression. This is to be scheduled at Day 14after T cell infusion for Cohort 2 subjects. The frequency of totaltumor-infiltrating lymphocytes and T cell subsets as well as theexpression of molecular markers of T cell antitumor response will bemeasured. These will be compared to baseline values on stored surgicaltumor specimens, if available. Subjects will be given the option torefuse tumor biopsy. Intracavitary fluid may also be submitted asbiopsy.

A mini-apheresis (2L) is done for research purposes and for FDA“lookback” purposes to archive CAR T cells for safety analysis at day 21post-infusion. A blood sample may be collected in lieu of leukapheresis.

Tumor Response Assessment

Tumor response will be assessed at Day 35 and Month 2 for Cohort 1 andDay 35, Months 2 and 6 following the last CAR T cell infusion for Cohort2 (and Extended Cohort 1), or until the patient requires alternative MPMtherapy. Tumor assessment will be done by CT scan with contrast forchest and other procedures as clinically indicated.

The above example can be done using human bearing scFv CARTs, using forexample the scFvs in CAR constructs M5, M11, M14, M15, M16 and M17.

Example 6: CART-MSLN Induce Anti-Tumor Activity in Solid Malignancies

This example present two case reports from ongoing clinical trialsindicating that adoptive transfer of mRNA CAR T cells (PBMCs wereelectroporated with mesothelin CAR construct) that target mesothelin(CART-MSLN) is feasible and safe without overt evidence of off-tumoron-target toxicity against normal tissues. These cases also demonstratethat CART-MSLN can infiltrate and have anti-tumor activity in solidmalignancies, such as malignant plural mesothelioma (MPM) and pancreaticcancer.

Subject 17510-105 had MPM and was given 3 infusions of CARTmeso. Subject21211-101 had metastatic pancreatic cancer (PDA) and was given 8 dosesof CARTmeso by intravenous infusion and 2 intratumoral injections. Bothpatients were elderly and had advancted chemotherapy-refractory cancerwith extensive tumor burden at the time of enrollment.

Anti-Tumor Clinical Activity of CART Meso Cells

Both cases were evaluated for tumor response by computed tomography (CT)imagining. In addition, the PDA patient was evaluated by[18F]2-fluoro-2-deoxy-D-glucose (FDG) avidity on positron emissiontomography/computed tomography (PET/CT) imaging before and afterinfusions (FIG. 6) The MPM patient had stable disease after receivingCARTmeso infusions, however, developed a confirmed partial responseafter reciving one infusion of CARTmeso cells on Schedule 2 (FIG. 6A).The PDA patient had stable disease after 3 weeks of intravenous CARTmesotherapy. By FDG PET/Ct imaging, a decrease in the maximum standardizeduptake value (SUVmax) was seen in all sites of disease. To further thismetabolic response, changes in the mean volumetric product (MVPmean) foreach disease site were determined (FIGS. 6B, 6C, and 6D). A decrease inMVPmean was observed only in the peritoneal lesions. To furtherunderstand the impact of CARTmeso cell therapy on peritoneal tumorburden, ascites fluid was analysed by flow cytometry. Analysis of theascitic fluid on days +3 and +15 after beginning therapy revealed a 40%decrease in the concentration of tumor cells that co-expressedmesothelin and c-met. (FIG. 6D). Overall, these findings suggest a rolefor CARTmeso cell infusions in inducing an anti-tumor effect in thesepatients.

Serological tumor markers was also evaluated in both patients. Serummesothelin-related peptide (SMRP) and CA19-9 levels were measured pre-and post-CARTmeso cell infusions. For the MPM patient, SMRP levelsdeclined afte receiving the first infusion on Schedule 2, coincidingwith the reduction of tumor burden seen by CT imaging. For the PDApatient, CA19-9 levels increased slowly over the course of treatment butremained stable for 1 month. After completion of the intra-tumoralinjections of CARTmeso cells, CA19-9 levels rose consistent with diseaseprogression.

In Vivo Persistence and Trafficking of CARTmeso Cells

A qPCR assay was develed to detect and quantify the persistence ofCARTmeso cells in patients following infusion. Analysis of peripheralblood, ascites, and tumor samples from the two patients are presented inFIG. 7. CARTmeso transgene was detected in both paitent immediatelyafter each infusion. In agreement with the biodegradable nature of theCARTmeso transgene, levels were observed to progressively decrease onsuccessive days.

Trafficking of CARTmeso cells in tumor tissues was evaluated in the PDApatient by collecting ascites at serial time point and by tumor biopsy.

Induction of Humoral Epitope Spreading after CARTmeso Cell Infusion

The hypothesis that CARTmeso cells, if able to recognize and lyseprimary tumor cells in vivo, might elicit a systemic anti-tumor immuneresponse was tested. High-throughput serological analysis was performedto measure induction of antibody responses to antigens to detect thedevelopment of a polyclonal immune response that may have occurred as aresult of tumor destruction and epitope spreading. These analysis wereaccomplished using an unbiased interrogation of treatment-induced igGresponses to almost 10,000 independent human proteins. In PDA patient,new antibody responses were detected at day +44 to more than 100proteins. Similarly, elevated antibody responses to a subset of proteinswas observed for the MPM patient. Overall, these antibody immuneresponses observed in both patients are consistent with CAR Tcell-mediated tumor destruction leading to the release of self-proteinsthat are cross-presented in a classical process of epitope spreading.

Pre- and post-treatment sera of both patients for the induction ofanti-tumor immune responses was also examined by immunoblotting usingpurified tumor associated proteins or protein lysates from human MPM orPDA cell lines. Anti-tumor immune responses were defined by the presenceof new bands or increases in the intensity of pre-existing bands onimmunoblots (FIG. 9) In both patients, antibody pattern alterations wereseen during treatment, e.g., increases in antibodies detecting proteinspresent in allogeneic tumor cell lines. These findings along with theprotoarray analysis suggest that an antitumor humoral immune responsewas induced by CARTmeso cell infusion.

Example 7: Combination Treatment to Improve CART Function in SolidTumors

Immunotherapy using adoptively transferred tumor infiltratinglymphocytes (TILs) is limited by T cell functional inactivation withinthe solid tumor microenvironment. In this study, human T cellsexpressing meso CAR were injected intravensously into immunodeficientmice bearing large, established human mesothelin-expressing mesotheliomaflank tumors. Analysis of the isolated meso-CAR T cells (mesoCART)showed rapid loss of effector functions that appeared to bemultifactorial. The results from these experiments demonstrate thatinhibitors of mechanisms that cause loss of effector function ofmesoCART may be useful to in a combination therapy to enhance efficacyof mesoCART treatment in vivo.

Materials and Methods

Generation of mesoCAR and lentivirus vector preparation. Lentiviralconstructs containing mesothelin CAR ss1 were prepared as describedpreviously. Human mesothelin CAR constructs and T cells expressing thesame can also be generated using methods previously described.

Cell lines. A human mesothelioma cell line derived from a patient'stumor was used—EMP (parental). Since EMP did not have baselineexpression of the tumor-associated antigen (TAA) mesothelin, it wastransfected with a lentivirus to stably express human mesothelin (thetransduced cell line was named EMMESO). Cells were also transduced tostably express firefly luciferase (called EMPffluc, EMMESOffluc).

T cell Effector Assays. Effector T cells were cocultured with fireflyluciferase expressing tumor cells at different ratios for a specifiedperiod time. At the end of the co-culture incubation period, supernatantwas saved for IFN levels by ELISA (Biolegend #430106), wells werewashed, and remaining tumor cells were lysed with 1× cell lysis bufferfor 30 minutes. The luciferase activity in the lysates was analyzedusing the Luciferase Assay System on a GloMax Multi Detection System(Promega.) Results are reported as percent killing based on luciferaseactivity in wells with tumor, but no T cells. (% killing=100-((RLU fromwell with effector and target cell coculture)/(RLU from well with targetcells)×100)). Effector-to-target ratios represent total T cells pertarget cell.

Animals. All animal experiments were approved by the appropriateInstitutional Animal Care and Use Committee. NOD/scid/IL2ry−/− (NSG)mice were bred in the Animal Services Unit of the Wistar Institute andChildren's Hospital of Philadelphia. Female mice were used forexperiments at 10 to 16 weeks of age.

In Vivo Xenograft Experiments. 5×10⁶ EMMESO tumor cells were injected inthe flanks of NSG mice in a solution of X-Vivo media (Lonza) andMatrigel (BD Biosciences). After tumors were established (100-200 mm³),the mice were randomly assigned to one of three intravenous (tail-vein)treatment groups: 1) 20×10⁶ non-transduced (NT) T cells (Dynabead®activated T cells), 2) 20×10⁶ mesoCAR T cells (Dynabead® activated Tcells transduced with mesoCAR) 3) saline. Tumors were measured usingcalipers and tumor volume was calculated using the formula(it/6)*(length)*(width)². Groups contained 10 mice each. The in vivoexperiments were repeated three times.

Results

Hypofunction Observed in Tumor Infiltrating CARTs

The human mesothelin-expressing mesothelioma tumor cell line EMMESO wasinjected into the flanks of NSG mice and allowed to grow to a sizebetween 100 and 200 mm3. At that time, tumor-bearing mice were given oneintravenous injection of 20 million T cells (mesoCAR expression wasapproximately 50% (data not shown)). Significant tumor growth slowingwas seen after a delay 14 days (FIG. 10), however, unlike our experiencewith another mesothelioma cell line (Carpenito et al., Proc Natl AcadSci USA, 106(9):3360-65 (2009), Zhao et al., Cancer Res, 70(22):9053-61(2010).) no tumor regression or cures were noted. Injection ofnon-transduced T cells had minimal anti-tumor effects when compared tothe saline treated control (FIG. 10), indicating that the reduction intumor growth observed in animals treated with T cells expressing mesoCARwas the result of the mesoCAR.

Various analyses were performed to understand why tumor regressions werenot observed after CART treatment. EMMESCO tumors were examined andmesoCARTs were found to be present in large numbers, however, the Tcells present had become hypofunctional. Given that the level of CARexpression on the CAR TILs was equal or greater to that of the cellsprior to injection (FIGS. 11A and 11B), we compared their functionalactivity. We isolated and analyzed the mesoCAR TILs from EMMESO tumors40 days after injection (all studies were started immediately afterisolation) and compared them to the same batch of mesoCAR T cells thathad been used for the original injection and frozen away (“cryomesoCAR”). These cells were studied after thawing and incubating in 37°C./5% CO2 for 18 hrs. When cryo mesoCAR T cells and flank mesoCAR TILswere added to cultured EMMESO cells expressing firefly luciferase(EMMESOffluc) at a 20:1 ratio for 18 hours, the cryo mesoCAR T cellswere highly efficient in killing tumor cells (>95%), while the mesoCARTILs killed only about 10% of the tumor cells over this same time period(FIG. 11C, p<0.001).

Additional analysis also demonstrated that the mesoCAR T cells did notproduce cytokines, such as IL-2 and IFNg, after exposure to antigen orPMA/Ionomycin. In contrast, the cryo-meso CAR T cells producedcytokines. The mesoCAR T cells also demonstrated reduced signaling, byreduced or absence of phosph-ERK expression by immunoblotting 20 minutesafter exposure to anti-CD3/CD28-coated beads. In contrast, cryo-meso CART cells retained phosphor-ERK expression after exposure to the beads.

Human mesoCAR TILs Express Increased Levels of Inhibitory Receptors

Next, the expression of four inhibitory receptors (IR) that have beenpreviously described in hypofunctional TILs isolated from humans wasevaluated using flow cytometry on: i) the cryo mesoCAR T cells that wereused for injection, ii) freshly isolated mesoCAR TILs from Day 39 fromEMMESO, and iii) “recovered” mesoCAR TILs that had been removed from thetumor for 24 hours (Table 6). CAR TILs expressed high levels ofinhibitory receptors. These levels were generally much lower after 24hours of recovery away from the tumor microenvironment. For the CD4 CARTILs, PD-1 went from 73% to 53%, LAG-3 went from 63% to 3%, and TIM3went from 24% to 1%. 2B4 expression was high and remained elevated afterrest (67% to 88%). For the CD8 CAR TILs, PD-1 went from 26% to 21%,LAG-3 went from 48% to 13%, and TIM3 went from 56% to 1%. 2B4 expressionwas high and remained elevated after rest (96% to 98%).

TABLE 6 Expression of inhibitor receptors (IRs) on TILs Percent positiveCD4 T cells Percent positive CD8 T cells Cyro Cyro Meso Meso EMMESO CART Fresh Recovered CAR T Fresh Recovered TIL cells TIL TIL cells TIL TILPD-1 37.2 73.0 53.0  5.4 26.0 21.0 (947) (1492) (1081) (326) (420) (360)LAG-3 44.7 62.8  3.0 85.2 48.0 13.0 (2585) (3845) (1968) (4609) (4376)(3161) 2B4  4.5 67.0 88.0 60.0 96.0 98.0 (215) (1102) (351) (2974)(6303) (5917) TIM3  1.9 24.0  1.0  2.4 56.0  1.0 (945) (1253) (200)(941) (1408) (435)

Three of these IR's were also evaluated on the human T cells that couldbe isolated from the spleens of the EMMESO mice. Interestingly, theexpression levels of PD-1, TIM3, and LAG3 were all lower on the splenicT cells compared with the TILs (Table 7), supporting a key role for thetumor microenvironment in the upregulation of IR's.

TABLE 7 Comparison of expression of inhibitor receptors on TILs andsplenic cells Percent positive Percent positive CD4 T cells CD8 T cellsEMMESO Fresh Splenic Fresh Splenic TIL TIL T cell TIL T cells PD-1 73 46(22) 26 15 (17) LAG-3 63 32 (132) 48 15 (11) TIM3 24 17 (9) 56 31 (8)

Human mesoCAR TILs Express Increased Levels of Intracellular InhibitoryEnzymes.

The expression levels of two intrinsic inhibitors of T cell functionthat have been implicated in TIL dysfunction, SHP-1 and DGK, wasexamined using immunoblotting (FIG. 12). The levels of both isoforms ofDGK (alpha and zeta), as well as the phosphorylated form of SHP1(pSHP1), were significantly elevated in mesoCAR TILs that were freshlyisolated from EMMESO flank tumor compared to overnight-rested TILs. Thiswas also confirmed for DGK using flow cytometry where 23% of freshEMMESO TILs expressed DGK. Expression was undetectable after overnightrest.

Block of Inhibitors in Human mesoCAR T Cells Enhances their Ex VivoKilling Function.

Given these expression data, the potential functional importance ofspecific inhibitory pathways in mesoCAR TILs was studied by introducingavailable blocking agents during the ex vivo killing and cytokinerelease assays. Addition of an anti-PDL1 antibody significantly restoredthe killing activity and ability to secrete IFNby the mesoCAR TILs(FIGS. 13A and 13B). The relatively high dose of 10 ug/ml anti-PDL1antibody was based on previously published investigations on cancerimmunotherapy. Addition of either a type I or type II DGK inhibitorsignificantly increased the killing ability (FIG. 13C), but withoutsignificantly increasing tumor-induced IFN secretion (FIG. 13D).Addition of the SHP1 inhibitor, sodium stibogluconate (SSG), slightlyinhibited the killing ability of cryo mesoCAR T cells, but significantlyincreased that of the mesoCAR TILs (FIG. 13E), as well as significantlyincreasing tumor-induced IFN secretion (FIG. 13F) of the mesoCAR TILs.Dose response curves were performed for both DGK inhibitors and for SSG,and the highest doses which did not induced direct tumor cell killingwere used.

Taken together, the results from these experiments demonstrate thathypofunction of CARTs in vivo is reversible. Specifically, modulation ofthe inhibitor mechanisms that reduce CART function can increase effectorfunction, and therefore, may increase therapeutic efficacy. Inhibitorsof inhibitor receptors, such as PD-1, LAG3, and TIM3, may be useful incombination therapy with CARTs for increased efficacy.

Example 8: Characterization of Human Meso CAR

Generation of Human CART-MSLN

PBMCs from a normal subject were isolated from a blood sample. PBMCswere transduced with lentivirus constructs containing the humanmesothelin CARs described herein. Transduction and culturing of thecells to produce CART-MSLN are described in the previous examples. TheCARTs produced express mesothelin CARS with scFv constructs includingM1, M2, M3, M4, M5, M6, M7, M8, M9, M10, M11, M12, M13, M14, M15, M16,M17, M18, M19, M20, M21, M22, M23, and M24. Control CARTs were alsogenerated, including CART expressing CAR-ss1 (murine anti-mesothelinscFv) (for positive control), and anti-CD19 CART (for negative control).

Cytokine Assay

Cytokine production after stimulation by tumor cells was assessed. CARTsexpressing M5, M11, M17, M21, ss1, and CAR19 were mixed with differenttumor cell lines at a 1:1 effector to target ratio, and plated at 50,000cell per well. The tumor cell lines were K562-meso (CML cell lineexpressing mesothelin), Ovcar3 (ovarian cancer), Ovcar8 (ovariancancer), and SW1990 (pancreatic adenocarcinoma). After 24 hours, CBAanalysis was performed to determine cytokine expression and/or secretionof IFNγ, TNF, IL-2, and IL-4.

As shown in FIG. 15A, CART-MSLN21 (M21) leads to the strongestactivation by K562-Meso. CART-MSLN5 and CART-MSLN11 were stimulated bySW1990 to produce IFNγ. The production/secretion of IL-2 was similar forthe tested CART-MSLN (FIG. 15C). As shown in FIG. 15D, there was hardlyany production of IL-4. Other studies also showed very little productionof IL-10.

Killing Assay

The ability for the CART-MSLN to target and kill tumor cells was alsoassessed in vitro. A luciferase-based assay was used, with tumor celllines expressing luciferase reporters. Target cells (tumor cells) wereplated at 20,000 targets/well. CART-MSLN were added at varyingeffector:target ratios, from 10:1, 5:1, 2.5:1, and 1:1. The cells werecultured for 20 hours, and the luminescence of each well was detected todetermine the percentage of target cells killed. Luciferase-expressingOvcar3 (ovarian cancer) and U87 mg (glioblastoma) cell lines were usedas target cells.

As shown in FIG. 16A, CARTs expressing M5, M11, M17 and ss1 performedsimilarly well in killing target tumor cells. No killing was observedfor CART-MSLN21. In contrast, no killing was observed for any of theCART constructs for glioblastoma cells (FIG. 16B). Similar results wereobtained for the panel of human meso CARs, where M11, M5, M17, and SS1had the highest specific killing of Ovcar3 cells (FIGS. 17A and 17B).

In Vivo Mouse Model

Anti-tumor activity of CART-MSLN was assessed in vivo in an Ovcar8xenograft model. 10×10⁶ Ovcar8 cells were implanted subcutaneously onday 0 in NSG mice. At day 14, 2×10⁶ CART cells (CARTs expressing M5,M11, M17, M21, SS1, and CD19, or untransduced cells; 10×10⁶ total Tcells) in a 100 μl dose was administered intravenously. Mice and tumorswere monitored for about 40 days after tumor cell implantation.

Results from this experiment show that CART expressing M5 and M11exhibit strong anti-tumor activity in this model (FIG. 18). Whencompared to the mock treated group, M5 and M11-treated mice exhibitedstatistically significant tumor reduction (p<0.05).

A second in vivo xenograft experiment was performed to assess theanti-tumor activity of a second set of CART-MSLN cells. 10×10⁶OVCAR8-mcherry cells were injected into NSG mice. Once tumors were150-250 mm², mice were randomized into treatment groups. CARTsexpressing M12, M14, M16, M23, ss1, CD19, or untreated cells wereinjected at a dose of 10×10⁶ T cells in 100 μl. About 40% of T cellswere expressing CAR, and therefore at the administered dosage, about4×10⁶ CART cells were delivered to each animal. Results from thisexperiment show that the CART expressing the human anti-MSLN (M12, M14,M16 and M23) did not have an effect on tumor growth (FIG. 19). Theseresults taken together with the results from the first set of CART-MSLNassessed indicate that M5 and M11 have specific anti-tumor activity incomparison with the other CART-MSLN tested, and thereby demonstratetheir potential for further investigation for cancer therapy. Inaddition, CART-SS1 did not exhibit any anti-tumor activity in the firstxenograft or the second xenograft experiment, further indicating that M5and M11 containing CART-MSLN are more effective at reducing tumorgrowth.

Example 9: DGK Inhibition Augments CART Efficacy

Previous studies, for example, the experiments discussed in Example 6,have suggested that CAR T cells lose efficacy over time in vivo (e.g.,in the tumor microenvironment). Specifically, mesoCAR T cells that wereinjected into a tumor mouse model were isolated from tumors after T cellinfusion (e.g., 39 days after, hereinafter referred to as tumorinfiltrating lymphocytes, TILs) and were assessed for their functionalactivity in comparison to freshly thawed mesoCAR T cells. The resultsshowed that in ex vivo killing assays and IFNγ release assays, themesoCAR T cells isolated from the tumor had reduced ability to killtumor cells (FIG. 20A), reduced IFNg production (FIG. 20B), and reducedERK signaling (as shown by phosphorylation in western blot analysis,FIG. 20C) in response to antigen or CD3/CD28 stimuli (indicating reducedT cell activation.

Inhibitory mechanisms that possibly explain the decrease in CAR T cellactivity in vivo over time include: soluble factors (TGFb, PGE2,adenosine, IL10, RAGE ligands, etc.), cell to cell contact (PD-1, LAG3,CTLA4, TIM3, CD160, etc.), and intrinsic activation-inducedintracellular negative feedback systems (diaglycerol kinases: a andisoforms, Egrs (2 and 3), SHP-1, NFAT2, BLIMP-1, Itch, GRAIL, Cbl-b,Ikaros, etc.). T cell activation can induce factors such as DGK. DGK, inturn, inhibits DAG signaling by phosphorylating DAG. This limitsDAG-induced activation of the RAS-ERK-AP1 pathway that leads to T cellactivation. Previous studies have shown that mice deficient in DGKa orDGK results in CD4 T cells that demonstrate enhanced signal transductionand appear more resistant to anergy-inducing stimuli.

In Vitro Cyotoxicity and Cytokine Release Assays

To investigate the effect of DGK inhibition on CART cell efficacy,transgenic mice with deletions in DGK genes DGKa, DGK, or both wereutilized. Splenic T cells from wild-type and DGK-deficient mice wereisolated, and transduced to express mesoCAR (SS1 BBZ) using retrovirus.MIGR1 CAR was used as a control.

A cytotoxicity assay was performed using similar methods to thosedescribed in previous Examples. Wild-type and DGK-deficient (KO) mesoCARexpressing cells were incubated at various effector:target ratios andcytotoxicity (% of target cells killed) was quantified (FIG. 21). Asshown in FIG. 21, deletion of DGKs markedly enhanced effector functionof CAR T cells, especially at low effector: target ratios.

Similarly, IFNγ release was examined in response to target cells atvarying effector:target ratios after 18 hours. As shown in FIG. 22,deletion of DGKs was found to markedly enhance efefctor function of themesoCAR T cells, especially at low effector:target ratios.

Western blot analysis of DGK-deficient mesoCAR T cells in comparison towild-type mesoCAR T cells showed increased ERK phosphorylation (FIG.23), indicating that presence of DGK suppresses ERK signaling, whiledeletion of DGK results in increased ERK signaling. The increase in ERKsignaling in the DGK deleted background suggests that inhibition of DGKresults in activation of the Ras-ERK-AP1 pathway, and therefore, T cellactivation.

Recent studies have shown that TGFβ modulates the functionality oftumor-infiltrating CD8 T cells though interfering with RAS/ERK signaltransduction, the same signaling molecules by which DGK deficiencyconfers augmented T cell effects. Sensitivity of the DGK-deficientmesoCAR T cells to TGFβ was examined. WT and DGK-deficient mesoCAR Tcells was incubated with mesothelin-expressing AE17 tumor cells + or −10ng/ml of TGFβ for 18 hours. Cytotoxicity and IFNg production by these Tcells was measured. As shown in FIG. 24, TGFβ inhibited killing by 50%in WT CAR T cells (arrows). However, this TGFβ-induced inhibition wasnot observed in CAR T cells with DGK deletion, demonstrating thatDGK-deficient cells are not sensitive to TGFβ modulation, and are moreresistant to inhibitor stimuli such as TGFβ, which may contribute to theincrease in T cell activity.

Therapeutic Efficacy of mesoCAR and DGK Inhibition In Vivo

Next, therapeutic efficacy of mesoCAR T cells was examined in thecontext of DGK inhibition or deficiency. AE17meso tumor cells(mesothelioma cells) were injected subcutaneously into C57BL/6 mice.When tumors reached 100 mm³ (approximately a week later), 10 millionmesoCAR T cells were injected intravenously via tail vein. Tumor volumeswere then followed over at least 18 days.

DGK-deficient mesoCAR T cells demonstrated enhanced and prolongedanti-tumor activity compared to wild-type (WT) mesoCAR and untreatedcells (FIG. 25A). Specifically, each of the three DGK-deficient mesoCART cells was shown to inhibit tumor growth by volume compared to WT anduntreated cells up to 18 days after injection. DGKz-deficient cellsexpressing mesoCAR were also shown to persist and proliferate betterthan wild-type meso CAR T cells in mice (FIG. 25B).

These results taken together show that DGK inhibition in combinationwith mesoCAR T cell treatment can improve mesoCAR T cell activation andanti-tumor activity in therapy.

Example 10: Inhibition of Ikaros Augments Anti-Tumor Capacity of CAR-TCells

One of the major hurdles in CART cell therapy is up-regulation ofintrinsic negative regulators of T cell signaling, such asdiacylglycerol kinase (DGK). As described in Example 9, CAR T cells havebeen shown to lose efficacy in vivo over time. Inhibition of negativeregulators of T cell function such as DGK was shown to enhance activityand function of CAR-expressing T cells.

Another important negative regulator of T cell function is thetranscription factor Ikaros. Unlike DGKs which act mainly in proximalTCR signaling, Ikaros is a zinc finger DNA binding protein thatnegatively regulates gene expression through the recruitment ofchromatin remodeling complexes, such as Sin3A, CtBP, and HDACs. Ikarosplays a role in regulating cytokine production and cytolytic function inCD4+ T cells and CD8+ T cells,

In this example, anti-tumor efficacy of retrovirally-transduced CAR Tcells with reduced Ikaros expression was examined in vitro and in vivo.

Materials and Methods

Cell lines. Mouse AE17 mesothelioma cells were described in Jackman etal., J Immunol. 2003; 171:5051-63). Human mesothelin were introducedinto AE17 cells by lentiviral transduction. 3T3Balb/C cells, werepurchased from the American Type Culture Collection. Mouse FAPexpressing 3T3BALB/C (3T3.FAP) cells were created by lentiviraltransduction of the FAP-3T3 parental line with murine FAP.

Animals. Pathogen-free C57BL/6 mice were purchased from Charles RiverLaboratories Inc. (Wilmington, Mass.). Ikaros DN+/− mice contain onewildtype Ikaros allele and one Ikaros allele with a deletion of a DNAbinding domain (Winandy et al., Cell. 1995; 83:289-99). Ikzf1+/− micehave one wildtype Ikaros allele and one allele with deletion of exon 7(Avitahl et al., Immunity. 1999; 10:333-43). Animals used for allexperiments were female mice between 6 and 12 weeks old and were housedin pathogen-free animal facilities.

Isolation, Transduction and Expansion of Primary Mouse T lymphocytes.Primary murine splenic T cells were isolated using the “Pan T cellNegative Selection” kit as suggested by the manufacturer (MiltenyiBiotec), and activated in 24-well plates (4×10⁶ cells/well in 2 mLsupplemented RPMI-1640 with 100 U/mL IL-2) pre-coated with —CD3 (1μg/mL) and —CD28 (2 μg/mL). After 48 hours, cells (1×10⁶ cells/well)were mixed with retrovirus (1 mL crude viral supernatant) in a 24-wellplate coated withRetronectin (50 μg/mL; Clontech) and centrifuged,without braking, at room temperature for 45 minutes at 1200 g. Afterovernight incubation, cells were expanded with 50 U/mL of IL-2 foradditional 48 hours.

Antigen- or antibody-coated beads. Recombinant mesothelin-extracellulardomain protein, bovine serum albumin (Fisher Scientific) oranti-CD3/anti-CD28 antibodies (eBioscience) were chemically crosslinkedto tosylactivated 4.5 μm Dynabeads (Invitrogen, #140-13) permanufacturers' instructions.

Immunoblotting. Anti-mesothelin-CAR transduced T cells were incubatedeither with BSA-, mesothelin-, or anti-CD3 antibody-coated beads (at 2:1bead to T cell ratio) for 5 and 20 min. Total cell lysates were thenprepared and immunoblotted for phosphorylated ERK, phosphorylated AKT,phosphorylated IKK, phosphorylated JNK, phosphorylated Lck,phosphorylated PKC, phosphorylated PLC, or phosphorylated ZAP70. Allanti-phospho-protein antibodies were purchased from Cell Signaling, withexception of anti-phospho-Lck, which was purchased from Sigma Aldrich. AC-terminus reactive goat anti-mouse antibody to Ikaros (SC-9861) and agoat anti-mouse actin antibody (SC-1615) were purchased from Santa Cruz.β-actin expression levels were determined to normalize the differencesin loading.

Cytotoxicity and IFN ELISA. AE17, AE17.meso, 3T3 and 3T3.FAP cells weretransduced with luciferase as described (Moon et al., Clinical CancerResearch. 2011; 17:4719-30). T cells and target cells were co-culturedat the indicated ratios, in triplicate, in 96-well round bottom plates.After 18 hours, the culture supernatants were collected for IFN analysisusing an ELISA (mouse IFN, BDOpEIA). Cytotoxicity of transduced T cellswas determined by detecting the remaining luciferase activity from thecell lysate using a previously described assay (Riese et al., CancerRes. 2013; 73:3566-77).

CAR T cell transfer into mice bearing established tumors. Mice wereinjected subcutaneously with 2×10⁶ AE17.meso tumor cells into thedorsal-lateral flank of C57BL/6 mice. Mice bearing large establishedtumors (100-150 mm³) were randomly assigned to receive either wildtypeCAR T cells, Ikaros-deficient CAR T cells or remained untreated(minimum, five mice per group, each experiment repeated at least once).1×10⁷ T cells were administered through the tail vein. Tumor size wasmeasured by electronic scales and calipers, respectively.

For Day 9 T cell activity assessment, spleen and tumors were processedinto single cell suspensions as previously described (Moon et al.,Clinical Cancer Research. 2014; 20(16):4262-73). Splenocytes and tumorsingle cell suspensions were re-stimulated with soluble anti-CD3/CD28antibodies (1.0 μg/ml) or with phorbol ester/ionomycin (PMA/I: 30 ng/ml,1 uM) for 4-6 hours in the presence of Golgi Stop (BD Biosciences, 0.66μl/ml) and then harvested for flow cytometric analysis.

Flow cytometric assays. Fluorochrome conjugated antibodies againstanti-mouse IFN-γ (XMG1), anti-mouse CD25 (PC61), anti-mouse IL-2(JES6-1A12), anti-mouse CD8 (53-6.7), anti-mouse CD44 (IM7), andanti-mouse CD4 (GK1.5) were purchased from Biolegend. Fixable, Live/DeadAqua stain (L34957) was purchased from Invitrogen. Fluorochrome antibodyto anti-mouse Granzyme B (NGZB) and FoxP3 (FJK-16s) was purchased fromeBioscience. Fluorochrome antibodies to anti-mouse TNF-α (MP6-XT22) andanti-mouse CD69 (H1.2F3) were purchased from BD Biosciences. Forintracellular cytokine staining, cells were treated with Golgi Stop (BDBiosciences, 0.66 μg/ml) for 4-6 hours. Following harvesting, cells werefixed with 1% paraformaldehyde for 30 minutes, spun down and washed oncewith FACS buffer. Cells were then washed with BD Perm Wash (BDBiosciences) 2 times and then stained with cytokine antibodies for 45minutes at room temperature. Cells were washed 2 times in BD Perm Washand then re-suspended in FACS Buffer. For transcription factor staining,cells were surfaced stained with fluorochrome-labeled primary antibodiesfor 20 minutes on ice. After washing in FACS buffer, cells were fixedwith Fix/Perm buffer from eBioscience. Following fixation, cells werepermeabilized and stained with APC anti-mouse FoxP3. For Ikarosstaining, rabbit anti-mouse Ikaros (Abcam, ab26083, 1:2000) was usedfollowing fixation and permeabilization with the eBioscience FoxP3 kit.Following staining with the Ikaros antibody, cells were washed and thenstained with a PE-labeled anti-rabbit secondary antibody (1:2000).Following completion of stains, cells were processed on a CyanADP(Beckman Coulter) for flow cytometric analysis.

Statistical Analysis. All statistical tests were done with GraphPadPrism. Two-way ANOVA was conducted with post-hoc testing, with *p<0.05,**p<0.01, ***p<0.001, and **** p<0.0001. Data are presented asmean+/−SEM.

Results

Cytokine Production and Cytolytic Mediator Release in CAR-Expressing TCells with Reduced Levels of Ikaros are Augmented

Given that cytolytic T lymphocytes (CTLs) with reduced Ikaros haveenhanced effector function in vitro and in vivo (O'Brien, et al., JImmunol. 2014; 192:5118-29), experiments were performed to test ifdepletion of Ikaros could improve the efficacy of CAR T therapy. T cellsisolated from wild type C57BL/6 and Ikaros-haplodeficient mice(Ikzf1+/−) in the C57BL/6 background were retrovirally-transduced toexpress a mesothelin CAR. Following ex vivo activation, transduction,and expansion in IL2, it was confirmed that, in comparison to wild-type(WT) CAR T cells, Ikzf1+/−CAR T cells continued to express less Ikarosprotein by flow cytometry and western blot (FIG. 26A).

Since Ikaros is a transcriptional repressor for multi-cytokine gene loci(Thomas et al., J Immunol. 2007; 179:7305-15; Bandyopadhyay et al.,Blood. 2006; 109:2878-2886; Thomas et al., J of Biological Chemistry.2010; 285:2545-53; and O'Brien et al., J Immunol. 2014; 192:5118-29), itwas next examined if reduction of Ikaros resulted in autocrine cytokineproduction by CAR T cells and also whether or not theIkaros-haplodeficient CAR T cells responded better than their WTcounterparts to their target antigen. Both WT and Ikzf1+/−CAR T cellswere stimulated with beads coated with either BSA-(control) ormesothelin (the CAR antigen) at a 2:1 bead:T cell ratio for 6 hours, andanalyzed their ability to produce IFNγ, TNFβ and IL2 by flow cytometry.At baseline (BSA stimulation), there was an ˜3-fold increase inIFN-producing Ikzf1+/−CART cells compared to WT CART cells (4.35% vs1.4%, FIG. 26B), but there was no significant difference in the % IL2producing cells (FIG. 26D). Following stimulation with mesothelin-coatedbeads, there was a dramatic increase in the % IFN-γ cytokine-producingIkzf1+/−CAR T cells (25%) while the response was modest in WT CAR Tcells (7%). An increase in TNFα production was also seen (FIG. 26C). Toinvestigate if this augmentation in cytokine production was generalizedacross different stimuli or limited to CAR antigen, both WT andIkzf+/−CAR T cells were treated with PMA and ionomycin for 6 hours. Inthis case, more IFN γ, TNF-β and IL-2 cytokine-producing cells wereobserved in the Ikzf1+/−CAR T cell compared to their WT counterparts(FIGS. 26B-26D). These data support the hypothesis that Ikaros is one ofthe limiting factors that suppresses cytokine production of T cells, orCAR T cells.

An important cytotoxic mediator, granzyme B, was shown to beup-regulated in CD3/CD28-activated Ikaros-deficient OT-I cells, and thisincreased their cytolytic activity against OVA-expressing EL4 tumorcells (O'Brien et al., J Immunol. 2014; 192:5118-29). It washypothesized that granzyme B production would also be enhanced inIkzf+/−T cells bearing CAR. Both WT and Ikzf1+/−CAR T cells werestimulated with either BSA-(baseline) or mesothelin-(CAR antigen) coatedbeads at 2:1 bead:T cell ratio for 6 hours. PMA/ionomycin was used asthe positive control for the assay. Similar to the data above, granzymeB level was higher in Ikzf+/− CAR T cells than in WT CAR T cells atbaseline (BSA stimulation; FIG. 26E). After stimulation with eithermesothelin-coated beads or PMA/Ionomycin, granzyme B level increased inboth WT and Ikzf+/−CAR T cells but the production was much higher in theIkzf+/− CAR T cells (FIG. 26E). To determine if there was also adifference in degranulation of CAR T cells with reduced Ikaros, CD107aexpression after antigen stimulation was assessed. Wild-type transducedT cells had moderate levels of CD107a expression following antigenre-stimulation, however, the T cells with reduced Ikaros demonstratedenhanced CD107a up-regulation (FIG. 26F). Thus, in response tore-stimulation, the CTLs with reduced Ikaros degranulate more andrelease more cytotoxic mediators in comparison to their wild-typecounterparts.

Depleting Ikaros with a Dominant Negative Allele Enhances CAR T CellFunction

In addition to the cells with lower levels of Ikaros, T cells from miceexpressing one dominant-negative allele of Ikaros (IkDN) were studied.Transgenic mice expressing IkDN have normal lymphoid development buthave peripheral T cells with 90% reduced Ikaros DNA binding activity(Thomas et al., J Immunol. 2007; 179:7305-15; and Winandy et al., Cell.1995; 83:289-99). T cells isolated from spleens of WT and IkDN mice wereactivated with plate-bound anti-CD3/CD28 antibodies, transduced withmesothelin CAR, followed by expansion with IL2. Knockdown of Ikaros inIkDN CAR T cells was confirmed by western. WT and IkDN CAR T cells werere-challenged with either BSA- or mesothelin-coated beads at 2:1 bead:Tcell ratio for 6 hours, and analyzed their ability to produce IFN andIL2, as well as to de-granulate in response to CAR antigen. Similar tothe Ikzf1+/− data above, some autocrine IFNγ production at baseline wasobserved (FIG. 27A), but not with IL2 (BSA stimulation; FIG. 27B). Uponligation of the CAR with its target antigen, mesothelin, IkDN T cellsmade more IFNγ than WT T cells (Mesothelin stimulation; FIG. 27A).De-granulation, as measured by CD107a up-regulation, was also similar inboth wild-type and IkDN CAR T cells (FIG. 27D).

Depletion of Ikaros Did not Augment Activation and Signaling of CAR TCells Following Antigen Stimulation

Given that depletion in Ikaros augmented cytokine release and increasedthe Granzyme B levels and CD107a expression of CART cells, possiblemechanisms were explored. It is plausible that these changes in effectorfunction could be due to differences in the activation of the wild-typeand Ikzf1+/− transduced T cells. Thus, the levels of CD69, CD25 and4-1BB (markers of T cell activation) were measured by flow cytometryafter stimulating with mesothelin-coated beads for 6 and 24 hours. CD69,an early activation marker, was up-regulated to the same extent by boththe wild-type and Ikzf1+/− cells (FIG. 28A). With longer stimulation,the wild-type and Ikzf1+/−CAR-expressing T cells continued to expresssimilar levels of CD69, but Ikzf1+/− transduced T cells exhibitedincreased CD25 expression (FIG. 28B). This may not directly indicate adifference in T cell activation, however, as increased IL-2 by Ikzf1+/−cells (FIG. 28D) can act in a positive feed-forward loop on CD25, theIL-2Ra (Depper et al., Proc Natl Acad of Sci USA. 1985; 82:4230-4; andNakajima et al., Immunity. 1997; 7:691-701). 4-1BB, a member of the TNFReceptor superfamily is also expressed on activated T cells (Vinay etal., Seminars in Immunology. 1998; 10:481-9) and was expressed atsimilar levels by CAR transduced wild-type and Ikzf1+/−T cells followingantigen stimulation (FIG. 28C). Thus, functional differences between ourWT and Ikzf1+/− transduced T cells were not due to differences in T cellactivation.

The experiments described in Example 9 and Riese et al., CancerResearch. 2013; 73:3566-77, demonstrate that depletion of the enzymediacylglycerol kinase (DGK) in CAR T cells resulted in an increase inRAS/ERK signaling, which correlated well with enhanced activation of CART cells. Some signaling pathways in WT and Ikaros-deficient T cellsafter TCR stimulation with CD3/CD28 antibodies were examined. Lysatesfrom stimulated T cells were prepared and immunoblotted for variousphospho-proteins implicated in proximal (PLC and Lck) and distal(ERK1/2, JNK, AKT and IKKα) signaling from the TCR. There was aconstitutive low-level baseline activation of some TCR signalingproteins in Ikaros-deficient T cells, including Lck, ERK and AKT (FIG.28D). With TCR/CD28 stimulation, all proteins studied werephosphorylated to the same level when comparing WT and Ikaros-deficientT cells, with the exception of phospho-IKK, which was slightly higher inIkaros-deficient T cells 20 minutes after stimulation. To determine ifthe NFB pathway was enhanced in T cells with reduced Ikaros level, thesame blot was re-probed for IB, the downstream target for IKK. There wasno difference in IB degradation between both WT and Ikaros-depleted Tcells. To study CAR signaling, both WT and IkDN mesothelin CARtransduced T cells were re-stimulated with mesothelin-coated beads.Similar to the data with CD3/CD28 stimulation, no difference was foundin phosphorylation of PLC and ERK (FIG. 28E). Together, these dataindicate that depletion in Ikaros does not alter TCR/CAR-mediatedsignaling.

Reduction of Ikaros in CAR T Cells Augments their Response Against theirTarget Cells.

Given the increased production of effector factors by CAR T cells withreduced Ikaros, their efficacy against their target tumor cells in vitrowas tested. Wild-type, Ikzf1+/− and IkDN T cells expressing mesoCAR weremixed at different ratios with the parental tumor cell line, AE17 or themesothelin-expressing cell line, AE17meso. When mixed with the parentalcell line, both the wild-type, Ikzf1+/− and IkDN T cells failed toproduce IFN-γ or lyse cells in response to AE17 (FIGS. 29A, 29B and29C). In contrast, when reacted with AE17meso, IFN-γ production andcytolysis by wild-type T cells increased as the E:T ratio increased(FIGS. 29B and 29C). However, both the Ikzf1+/− and IkDN T cellsproduced significantly more IFN-γ and had significantly increased tumorlysis than wild-type T cells, even at the lowest E:T ratio 1.3:1 (FIGS.29B, and 29C).

To study the generalizability of this effect, T cells expressing adifferent CAR construct, which targets fibroblast activation protein(FAP-CAR) and has the same intracellular signaling domain as themesothelin CAR used above, were examined. The efficacy of comparablytransduced FAP-CAR splenic T cells isolated from WT C57BL/6 was comparedto those from Ikzf1+/− mice. Ikzf1+/− FAP-CAR T cells were moreefficient in lysing 3T3.FAP cells (FIG. 29D) and in secreting more IFN(FIG. 29E) than WT FAP CAR T cells, with retention of specificity invitro.

Depletion of Ikaros Enhances the Efficacy of CAR T Cells AgainstEstablished Tumors

The capability of mesothelin-specific T cells with reduced Ikaros(Ikzf+/− and IkDN) to control growth of established AE17meso tumors inmice was next examined. Two million of AE17meso tumor cells wereinjected into the flanks of syngenic C57BL/6 mice and allowed to formlarge established tumors (−100-150 mm³). Ten million CAR T cellsprepared from WT or Ikaros-deficient (Ikzf+/− and IkDN) mice were thenadoptively transferred into those tumor-bearing mice, and tumormeasurements were followed. Mild tumor growth inhibition was induced bywild-type transduced mesoCAR T cells, while both Ikzf1+/− and IkDNtransduced mesoCAR T cells inhibited growth of AE17meso tumorssignificantly more (FIGS. 30A and 30B).

It was also studied if reduction of Ikaros could enhance the therapeuticpotential of FAP-CAR T cells. Mice with established AE17meso tumors(100-150 mm³) were adoptively transferred with 10 millions wild-type orIkzf+/− transduced anti-mouse FAP CAR T cells. Mice receiving wild-typetransduced cells provided minimal tumor delay and the AE17meso tumorscontinued to grow (FIG. 30C). In contrast, the Ikzf+/− transduced Tcells were able to significantly delay tumor growth.

Ikzf1+/−CAR T Cells Persist Longer and More Resistant toImmunosuppressive Tumor Microenvironment than WT CAR T Cells

Given the enhanced efficacy of the Ikaros-inhibited CAR T cells, thepossible mechanisms using the Ikzf1+/− mesoCAR T cells were explored. Tofurther interrogate how these mesoCAR T cells operate in animmunosuppressive tumor microenvironment in vivo, tumors at 3 and 9 dayspost-adoptive transfer were harvested and assessed their number andfunctionality. These two time points allowed characterization of theiractivity at early and late time points during the anti-tumor immuneresponse.

At Day 3 post-transfer, we observed a similar frequency of wild-type andIkzf1+/− mesoCAR T cells in both the spleens (FIG. 31A) and tumors (FIG.31B). These similar levels indicate that both the wild-type and Ikzf1+/−mesoCAR T cells initially traffic equally well to the tumor. Inassessing the Day 9 timepoint, the number of both wild-type mesoCAR Tcells Ikzf1+/− mesoCAR T cells declined in the spleen. However, when thetumors were examined at this time point, there was a significantincrease in the number of Ikzf1+/− mesoCAR T cells compared with WTmesoCAR T cells (FIG. 31B). These data show that the Ikzf1+/− mesoCAR Tcells either persist or proliferate better than WT mesoCAR T cells inthe immunosuppressive microenvironment.

Tumor infiltrating lymphocytes (TILs) become hypofunctional in responseto their cognate antigens within the immunosuppressive tumormicroenvironment, and is a key phenomenon associated with tumorprogression (Prinz et al., J Immunol. 2012; 188:5990-6000; and Kerkar etal., Cancer Res. 2012; 72:3125-30). Although there were more Ikzf1+/−mesoCAR T cells in the tumors at Day 9, they could still be adverselyaffected by the tumor microenvironment. To evaluate functionality,CD3/CD28 antibodies were used to stimulate TILS isolated from wild-typeand Ikzf1+/− mesoCAR T cells at Day 9 post-transfer and characterizeddifferences in lytic mediator production. At Day 9 post-transfer, thewild-type mesoCAR T cells in the spleen continued to produce somemoderate levels of IFN (FIG. 31C). As expected, isolated wild-type Tcells from the tumors produced much less of this cytokine in comparisonto wild-type T cells isolated from the spleen. This indicates that thewild-type TILs begin to become hypofunctional at Day 9 post transfer. Incontrast, splenic Ikzf1+/− mesoCAR T cells continued to produce moreIFN-at baseline and upon stimulation (FIG. 31C). Compared to thewild-type TILs, the Ikzf1+/− TILs produced higher amounts of IFNγ. Thesedata indicate that Ikzf1+/− TILs could be less sensitive to theimmunosuppressive tumor microenvironment.

Bypassing the proximal defect of TCR signaling often seen in TILs(Prinz, P U et al., Immunol. 2012; 188:5990-6000) can be achievedthrough use of PMA/Ionomycin (PMA/I) (Prinz et al., J Immunol. 2012;188:5990-6000). Wild-type and Ikzf1+/− mesoCAR T cells werere-stimulated with PMA/I to determine if TCR-stimulation insensitivewild-type and Ikzf1+/−TILs were still capable making cytokines inresponse to other stimuli. At Day 9 post-transfer, the wild-type TILsdemonstrated a noticeable drop in IFN-γ production (FIG. 31C), and thiswas partially restored through stimulation with PMA/I (FIG. 31D).However, stimulation of splenic and tumor isolated Ikzf1+/− mesoCAR Tcells still resulted in increased levels of IFN-γ in comparison towild-type transferred cells. Through bypassing any defects in TCRsignaling via PMA/I stimulation, these results demonstrate that IFNγproduction differs at the chromatin level and is likely due todifferential Ikaros function.

In light of the increased anti-tumor activity by the transferredIkzf1+/−T cells, the impacts on the composition of the immunosuppressivetumor microenvironment was also examined and the number of regulatory Tcells (Tregs) and Myeloid Derived Suppressor Cells (MDSCs) was evaluatedat Day 9. At Day 9 post-transfer, similar levels of Tregs in hosts thatreceived wild-type or Ikzf1+/− mesoCAR T cells was observed (FIG. 31E).The presence of Ly6G−/CD11b+/CD206+ macrophages was determined, whichare typically characterized as immunosuppressive and pro-tumorigenic M2macrophages. In the Day 9 treated groups, that CD206 expression wassimilar in all 3 groups (untreated, wild-type, and Ikzf1+/−) (FIG. 31F).

T Cells with Reduced Ikaros are Less Sensitive to Soluble InhibitoryFactors TGF and Adenosine

To further characterize the interaction of the immunosuppressive tumormicroenvironment with the mesoCAR T cells, an in vitro culture systemwas utilized. Soluble inhibitory factors such as IDO, IL-10, Adenosine,and TGF-β (Wang et al., Oncoimmunology. 2013; 2:e26492) have been shownto contribute to inhibiting infiltrating tumor lymphocytes. The effectsof select inhibitory factors in vitro on the Ikaros-deficient CAR Tcells was tested to determine if the immunosuppressive environment couldimpact their lytic function. Wild-type CAR T cells had a 50% reductionin their ability to make IFN-γ and had a reduction in their lyticfunction in the presence of TGF-β and Adenosine (FIG. 32). CAR T cellswith reduced levels of Ikaros (Ikzf+/− and IkDN) continued to producemore IFNγ than their wild-type counterparts in the absence of inhibitorsand were only marginally inhibited in the presence of TGF β andAdenosine (FIG. 32A). Increased lytic function in Ikzf+/− and IkDN CAR Tcells in comparison to wild-type T cells was observed (FIG. 32B). Thesedata demonstrate that the T cells with reduced Ikaros are less sensitiveto TGFβ and adenosine inhibition.

DISCUSSION

In this example, a new approach for enabling CAR-expressing T cells tosurvive and enhance their effector functions in the tumor environmenthas been identified: inactivation of the transcriptional repressorIkaros, which is known to inhibit a diverse array of genes involved in Tcell function, e.g., cytokine genes (IL2 and IFNγ), cytolytic mediators(granzyme B), and the key T-box transcription factors that influence Tcell differentiation (R-Bet and Eomes).

An important finding from the experiments described above is that CAR Tcells that were deficient in Ikaros function were significantly betterthan wild type CAR T cells in restricting tumor growth (FIG. 30). Theseresults were observed in multiple tumor models and using two differentCAR constructs. Due to the high number of genes that Ikaros regulates,it is plausible that Ikaros may regulate many pathways that are normallysensitive to immunosuppression. Increased IFN-g production by loweringIkaros level in CAR T cells can result in up-regulation of Class I MEWexpression on the tumor, and thereby improving its immunogenicity,improving anti-angiogenic activity, and driving STAT1 mediated fuctionof Thl cells. A possible effect of increased IFNγ could have been analternation of the macrophage phenotype within the tumors, however, nodifferences in the total number of macrophages, nor the proportion ofM2-like macrophages (as measured by CD206 expression) was observed. Theincreased IL-2 production could have also increased the formation of CD4Treg cells, however, no differences were observed when comparing thetumors treated with WT CAR T cells with Ikaros-deficient CAR T cells.

An increased number of tumor infiltrating lymphocytes nine days afterinjection was observed. This was not likely due to increasedtrafficking, since the number of WT versus Ikaros-deficient TIL wassimilar at Day 3. Instead, these results suggest that Ikaros-deficientTIL showed increased proliferation or decreased antigen-induced celldeath (AICD). In vitro studies suggest that AICD was similar between thetwo types of T cells, making it more likely that the difference was dueto increased proliferation. This would be consistent with the increasedIL-2 produced by these cells. In addition to increased persistence, theIkaros-deficient TIL appeared to be less hypofunctional. Whentumor-infiltrating CAR T cells were re-stimulated with anti-CD3 antibodyor PMA and ionomycin, CAR TILs with Ikaros deficiency were able to makemore IFNγ than their wild-type counterparts (FIGS. 31C and 31D).

The in vitro studies allowed further studies of the mechanisticunderpinnings of the observed increased anti-tumor efficacy in vivo.Consistent with the known inhibitor functions of Ikaros, deletion of oneIkaros allele (Ikzf+/−) or replacing one of its alleles with an Ikarosdominant negative construct (IkDN) resulted in T cells that had someincreased baseline autocrine IFNγ and Granzyme B production (FIG. 26),but more importantly, showed markedly augmented cytokine secretion andgranule release after TCR or CAR stimulation in vitro. This wasaccompanied by increased tumor cell killing in vitro. To test whether ornot Ikaros-deficient CAR T cells have lower activation threshold than WTCAR T cells, Dynabeads were coated with 10-fold less mesothelin proteinand it was shown that Ikzf+/−CART cells could still respond to themesothelin antigen at low-density to make IFNγ and TNF-α but the WT CART cells could not. The Ikaros-disabled CAR T cells were also moreresistant to inhibition by known immunosuppressive factors such as TGF-βand adenosine (FIG. 32). This may be due to the fact that cytokine (i.e.IL2 and IFNγ and T cell effector (i.e. granzyme B) genes are moreaccessible for transcription in Ikaros-deficient T cells followingTCR/CAR activation. This appears to help compensate for suboptimal Tcell activation within immunosuppressive tumor microenvironment.

In previous studies, e.g., Example 9, a similar phenotype (i.e. increasein cytolysis and IFN production) has been observed in CAR T cellsthrough depletion of DGKs, enzymes that metabolize the second messengerdiacylglycerol and limit RAS/ERK activation. With DGK deletion, however,clear changes were observed in the CAR/TCR signaling pathway.Specifically, RAS/ERK activation was dramatically enhanced after bothTCR and CAR activation. This resulted in enhanced activation, asmeasured by increased CD69 upregulation, however production of effectormolecules such as TRAIL, FasL and IFNγ. Perforin and Granzyme B weresimilar between WT and DGK-deficient CAR T cells. A very differentphenotype in this study with Ikaros-deficient CAR T cells. In contrastto the DGK-deficient CAR T cells, the Ikaros-deficient CAR T cells hadsimilar CAR/TCR activation as shown by CD69 and CD25 upregulation (FIG.28), and similar CAR/TCR signaling as measured by phosphorylation ofmultiple TCR signaling molecules (FIG. 34) and calcium signaling. UnlikeDGK-deficient T cells, Ikaros-depleted CAR T cells had higher granzyme Band IFN levels at baseline (FIGS. 26 and 27), as well as constitutivelow level activation of some TCR signaling cascades such as ERK and Akt(FIG. 28). This baseline activation may be due to induction of T-bet(Thomas et al., J of Biol Chemistry. 2010; 285:2545-53), whichcooperates with other transcription factor like Eomes to transactivateIFN and granzyme B gene expression (Pearce et al., Science. 2003;302:1041-3; and Intelkofer et al., Nat Immunol. 2005; 6:1236-44).

These findings raise the possibility that therapeutically targetingIkaros in transduced human T cells (in clinical trials) might bebeneficial using either genetic or biochemical approaches. Geneticapproaches could mimic these results in mice and include knockdown ofIkaros in CAR T cells using shRNA or use of a dominant negativeconstruct to compete with endogenous Ikaros. Another option would be touse a pharmacological inhibitor to lower Ikaros levels transiently.Recent reports have indicated that the immunomodulatory drug,Lenalidomide, targets Ikaros for ubiquitin-mediated degradation by theE3 ligase complex CRL4CRBN (Gandhi et al., Br J Haematol. 2013;164:811-21; Kronke et al, Science. 2014; 343:301-5; and Sakamaki et al.,Leukemia. 2013; 28:329-37). CD3-stimulated human T cells treated withLenalidomide produce more IL-2 (Gandhi et al., Br J Haematol. 2013;164:811-21), a key trait of T cells with reduced Ikaros levels. Inpreliminary studies, TCR/CD28 stimulated mesothelin-CAR transduced humanPBMCs in vitro produce more IL-2 and IFNγ after pretreatment withlenalidomide. Thus, the combination of CAR T cell therapy with in vivoadministration of Lenalidomide may provide a therapeutic strategy forreversal of T cell hypofunction through inhibition of Ikaros.

In conclusion, this example demonstrates for the first time thattargeting a transcriptional repressor can enhance CAR-mediatedanti-tumor immunity. The mechanisms involved enhanced cytokine andeffector function without alterations in signal transduction.Translating this approach into the clinic can be pursued through the useof shRNA, a dominant negative construct, or a pharmacological inhibitor(like Lenalidomide) to target Ikaros in CAR-expressing T cells.

Example 11: Meso-CART in Human Ovarian Cancer

In this example, the safety of intravenous infusion of autologous Tcells transduced to express a mesothelin CAR was determined in a humanpatient with advanced recurrent serou ovarian cancer.

A single patient treatment protocol was modeled after a planned Phase Istudy (as described in Example 3) to assess the safety and feasibilityof infusing autologous T cells expressing mesothelin CAR (mesoCART). Tcells from the patient were transduced to express a CAR comprising anextracellular anti-mesothelin single chain variable fragment (scFv)fused to 4-1BB and TCRzeta signaling domains. 3×10⁷ mesoCAR T cells/m²,for a total of 4.65×10⁷ meso CAR T cells, were infused into the patient.No lymphodepletion with chemotherapy was given prior to infusion.

The mesoCART infusion had no acute toxic effects. The patientexperienced grade 3 bilateral pleural effusions, dyspnea, fever,hypotension, and aspiration. There were no clinical toxicities due tooff tumor/on target effects of the meso CART cells, such as pericarditisor peritonitis. The patient was treated with tocilizumab (anti-IL6) onDay 21 after infusion for suspected cytokine release syndrome (CRS),based on unexplained fevers, hypotension requiring pressors, elevatedserum levels of ferritin (>7000 ng/mL) and CRP (>160 mg/L).

The mesoCAR T cells were detectable in peripheral blood samples, as wellas tumor samples from liver and peritoneum, with highest numbers inpericardial and pleural fluid. Cytokine evaluation showed a markedincrease in IL-6 within the pleural fluid (>80-fold increase overbaseline), peaking at Day 22-23. Evaluation of the blood also revealedelevation of IL-6 levels in the blood (15-fold increase over baseline atDay 22). No elevation in TNFα or IFNγ levels was detected.

Preliminary results showed that CT imaging of abdomen and pelvisperformed Day 21 showed peritoneal carcinomatosis minimally improvedcompared to a CT imaging two weeks prior. Specifically, one tumordecreased from 3.4×3.1 cm to 3.0×2.6 cm. Cytology from the left sidedpleural fluid showed malignancy on Day 8 after infusion, but at Day 21and Day 26, there was no evidence of malignancy.

These results showed that infusion of meso CART cells withoutlymphodepletion was found to be feasible and safe. There was no evidenceof toxicity with infusion, or life-threatening pericarditis orperitonitis. Also, there was clinical evidence of treatment efficacy asevidenced by the disappearance of malignant pleural effusions.

Example 12: Transient Expression of CARs with Varying IntracellularSignaling Domains

In this example, the use of distinct costimulatory domains in the CARarchitecture and its effects on cell longevity, memory differentiation,and cell metabolism characteristics are examined.

A novel system of in vitro T cell stimulation was developed to study theconsequences of a single round of CAR-specific stimulation and analyzesignaling through various CAR signaling endodomains. In vitrotranscribed mRNA encoding anti-mesothelin SS1-CAR constructs withvarying intracellular signaling domains of CD3zeta, CD28:z, and 4-1BB:zwere electroporated into primary resting human T cells. Greater than 90%CAR-positive T cell populations were achieved by this method. Uponverifying CAR expression, the T cells were stimulated with recombinantmesothelin immobilized on beads and then cultured. Since the RNA istransiently expressed, the CAR disappears from the cell surface afterone round of stimulation, allowing the unambiguous analysis of signalingthrough the CAR alone (i.e., without subsequent signaling fromadditional mesothelin binding events). This approach thus obviated therequirement for stimulation through the endogenous T cell receptor andpermitted for the first time an analysis of the consequences ofsignaling through the surrogate antigen of CAR T cells. Theseexperiments were conducted on primary peripheral blood T cells, sortednaïve T cells and cord blood cells.

The various CAR constructs (SS-1 with CD3zeta, SS1 with CD28:z, and SS1with 4-1BB:z) were expressed at equivalent levels on the surface of Tcells. All signaling domains were demonstrated to have comparable andspecific cytolytic capabilities when cultured with target cells thatexpressed mesothelin.

Primary T cells stimulated through the 4-1BBz-containing CARs showedsuperior survival and expansion profiles when compared to CD28-basedCARs. Both 4-1BB and CD28-based CARs exhibited more than 5 populationdoublings. However, the 4-1BB-based CAR T celsl proliferated for longerin vitro. Phenotypic analysis of the 4-1BB-based CAR T cells revealedthat an increased population of cells with central-memory surfacemarkers was generated. In contrast, the CD28-based CARs rapidlydifferentiated to an effector memory pool of CART cells.

CD28z-containing CARs yielded a significantly higher proportion ofeffector memory cells, with a modest increase in expression ofinhibitory PD-1, TIM3 and LAG3 molecules relative to their 4-1BBzcounterparts. These results remained consistent whether startingpopulations of bulk peripheral blood T cells, naïve(CD45RO-CD95-CD62L+CCR7+ sorted) peripheral blood T cells, or cord bloodT cells were used.

Metabolic profiling of the mesothelin-stimulated CAR T cells using theSea-Horse assay (Seahorse Biosciences) in culture revealed a substantialincrease in lipid oxidation in 4-1BBz-CAR stimulated cells compared totheir CD28z counterparts. Additionaly, microarray studies have revealeda unique gene signature in cells that are recovered after stimulationthrough the different CAR signaling domains.

Together, these results show that CAR T cells exhibit differentialsurvival and function depending on the costimulatory domains. Thissystem also allows for rapid characterization and functional analysis ofnew CAR designs.

Example 13: Effects of mTOR Inhibition on Immunosenescence in theElderly

One of the pathways most clearly linked to aging is the mTOR pathway.The mTOR inhibitor rapamycin has been shown to extend lifespan in miceand improve a variety of aging-related conditions in old mice (Harrison,D E et al. (2009) Nature 460:392-395; Wilkinson J E et al. (2012) AgingCell 11:675-682; and Flynn, J M et al. (2013) Aging Cell 12:851-862).Thus, these findings indicate that mTOR inhibitors may have beneficialeffects on aging and aging-related conditions in humans.

An age-related phenotype that can be studied in a short clinical trialtimeframe is immunosenescence. Immunosenescence is the decline in immunefunction that occurs in the elderly, leading to an increasedsusceptibility to infection and a decreased response to vaccination,including influenza vaccination. The decline in immune function with ageis due to an accumulation of immune defects, including a decrease in theability of hematopoietic stem cells (HSCs) to generate naïvelymphocytes, and an increase in the numbers of exhausted PD-1 positivelymphocytes that have defective responses to antigenic stimulation(Boraschi, D et al. (2013) Sci. Transl. Med.5:185 ps8; Lages, C S et al.(2010) Aging Cell 9:785-798; and Shimatani, K et al., (2009) Proc. Natl.Acad. Sci. USA 106:15807-15812). Studies in elderly mice showed that 6weeks of treatment with the mTOR inhibitor rapamycin rejuvenated HSCfunction leading to increased production of naïve lymphocytes, improvedresponse to influenza vaccination, and extended lifespan (Chen, C et al.(2009) Sci. Signal. 2:ra75).

To assess the effects of mTOR inhibition on human aging-relatedphenotypes and whether the mTOR inhibitor RAD001 amelioratesimmunosenescence, the response to influenza vaccine in elderlyvolunteers receiving RAD001 or placebo was evaluated. The findingspresented herein suggest that RAD001 enhanced the response to influenzavaccine in elderly volunteers at doses that were well tolerated. RAD001also reduced the percentage of programmed death (PD)-1 positive CD4 andCD8 T lymphocytes that accumulate with age. These results show that mTORinhibition has beneficial effects on immunosenescence in elderlyvolunteers.

As described herein, a 6 week treatment with the mTOR inhibitor RAD001,an analog of rapamycin, improved the response to influenza vaccinationin elderly human volunteers.

Methods Study Population

Elderly volunteers >=65 years of age without unstable underlying medicaldiseases were enrolled at 9 sites in New Zealand and Australia.Exclusion criteria at screening included hemoglobin <9.0 g/dL, whiteblood cell count <3,500/mm³, neutrophil count <2,000/mm³, or plateletcount <125,000/mm³, uncontrolled diabetes, unstable ischemic heartdisease, clinically significant underlying pulmonary disease, history ofan immunodeficiency or receiving immunosuppressive therapy, history ofcoagulopathy or medical condition requiring long-term anticoagulation,estimated glomerular filtration rate <30 ml/min, presence of severeuncontrolled hypercholesterolemia (>350 mg/dL, 9.1 mmol/L) orhypertriglyceridemia (>500 mg/dL, 5.6 mmol/L).

Baseline demographics between the treatment arms were similar (Table 8).Of the 218 subjects enrolled, 211 completed the study. Seven subjectswithdrew from the study. Five subjects withdrew due to adverse events(AEs), one subject withdrew consent, and one subject left the study as aresult of a protocol violation.

TABLE 8 Demographic and Baseline characteristics of the Study PatientsRAD001 RAD001 RAD001 0.5 mg 5 mg 20 mg Placebo daily weekly weeklypooled Total Population N = 53 N = 53 N = 53 N = 59 N = 218 Age (Years)Mean 70.8 72.0 71.4 71.1 71.3 (SD) (5.0) (5.3) (5.2) (5.1) (5.2) GenderMale-n   34   27   32   31  124 (64%) (51%) (60%) (53%) (57%) BMI* Mean27.4 28.8 28.0 28.0 28.0 (kg/m2) (SD) (4.2) (5.0) (4.1) (4.2) (4.4)Race-n (%) Caucasian   48   50   46   54  198 (91%) (94%) (87%) (92%)(91%) Other   5   3   7   5   20 (9%) (6%) (13%) (8%) (9%) *Thebody-mass index is weight in kilograms divided by the square of theheight in meters

Study Design and Conduct

From December 2011 to April 2012, 218 elderly volunteers were enrolledin a randomized, observer-blind, placebo-controlled trial. The subjectswere randomized to treatment arms using a validated automatedrandomization system with a ratio of RAD001 to placebo of 5:2 in eachtreatment arm. The treatment arms were:

RAD001 0.5 mg daily or placebo

RAD001 5 mg weekly or placebo

RAD001 20 mg weekly or placebo

The trial was observer-blind because the placebo in the RAD001 0.5 mgdaily and 20 mg weekly cohorts differed slightly from the RAD001 tabletsin those cohorts. The study personnel evaluating the subjects did notsee the study medication and therefore were fully blinded. The treatmentduration for all cohorts was 6 weeks during which time subjectsunderwent safety evaluations in the clinic every 2 weeks. After subjectshad been dosed for 4 weeks, RAD001 steady state levels were measuredpre-dose and at one hour post dose. After completing the 6 week courseof study drug, subjects were given a 2 week drug free break to reverseany possible RAD001-induced immunosuppression, and then were given a2012 seasonal influenza vaccination (Agrippal®, Novartis Vaccines andDiagnostics, Siena, Italy) containing the strains H1N1A/California/07/2009, H3N2 A/Victoria/210/2009, B/Brisbane/60/2008. Fourweeks after influenza vaccination, subjects had serum collected forinfluenza titer measurements. Antibody titers to the 3 influenza vaccinestrains as well as to 2 heterologous strains (A/H1N1 strain A/NewJersy/8/76 and A/H3N2 strain A/Victoria/361/11) were measured bystandard hemagglutination inhibition assay (Kendal, A P et al. (1982)Concepts and procedures for laboratory-based influenza surveillance.Atlanta: Centers for Disease Control and Prevention B17-B35). Levels ofIgG and IgM specific for the A/H1N1/California/07/2009 were measured inserum samples taken before and 4 weeks after influenza vaccination asdescribed previously (Spensieri, F. et al. (2013) Proc. Natl. Acad. Sci.USA 110:14330-14335). Results were expressed as fluorescence intensity.

All subjects provided written informed consent. The study was conductedin accordance with the principals of Good Clinical Practice and wasapproved by the appropriate ethics committees and regulatory agencies.

Safety

Adverse event assessment and blood collection for hematologic andbiochemical safety assessments were performed during study visits.Adverse event information was also collected in diaries that subjectsfilled out at home during the 6 weeks they were on study drug. Data onall adverse events were collected from the time of informed consentuntil 30 days after the last study visit. Events were classified by theinvestigators as mild, moderate or severe.

Statistical Analysis

The primary analysis of geometric mean titer ratios was done using anormal Bayesian regression model with non-informative priors. This modelwas fitted to each antibody titer on the log scale. The primary outcomein each model was the Day 84 measurement. The Day 63 measurement wasincluded in the outcome vector. The model fitted using SAS 9.2 procmixed with the prior statement. The covariance structure of the matrixwas considered as unstructured (option type=UN). A flat prior was used.For the secondary analysis of seroconversion rates, logistic regressionwas used.

The intention to treat population was defined as all subjects whoreceived at least one full dose of study drug and who had no majorprotocol deviations impacting efficacy data. 199 out of the total of 218subjects enrolled in the study were in the intention to treatpopulation.

Immunophenotyping

Peripheral blood mononuclear cells were isolated from whole bloodcollected at 3 time points: baseline; after 6 weeks of study drugtreatment; and at the end of study when subjects had been off study drugfor 6 weeks and 4 weeks after influenza vaccination. Seventy-six PBMCsubsets were analyzed by flow cytometry using 8-color immunophenotypingpanels at the Human Immune Monitoring Center at Stanford University, CA,USA as described previously (Maecker, H T et al. (2012) Nat Rev Immunol.12:191-200). Seventy-six PBMC subsets were analyzed by flow cytometryusing 8-color lyophilized immunophenotyping panels (BD Lyoplate, BDBiosciences, San Diego, Calif.). PBMC samples with viability >80% andyield of 2×10⁶ cells or greater were included in the analysis.

Relative changes of the immunophenotypes from baseline to Week 6 ofstudy drug treatment and from baseline to the end of study (Week 12)were calculated for each of the RAD001 dosing cohorts. Student T testwas conducted to examine if the relative change of the immunophenotypesfrom baseline to the two blood sampling time points was significantlydifferent from zero, respectively, within each dosing group afteradjusting for placebo effect. Missing data imputation in treatmenteffect analysis was not conducted. Therefore if a patient has a missingphenotype data at baseline, this patient was not be included in theanalysis for this phenotype. If a patient had a missing phenotype dataat 6 or 12 weeks, then this patient did not contribute to the analysisof this phenotype for the affected timepoint.

608 tests in 76 phenotypes under 3 dosing groups were conducted tocompare the treatment effect against the placebo effect. Stratifiedfalse discovery rate (FDR) control methodology was implemented tocontrol the occurrence of false positives associated with multipletesting yet provide considerably better power. The cell type group wastaken as the stratification factor and conducted FDR (q-value)calculation within each stratum respectively. All null-hypotheses wererejected at 0.05 significance level with corresponding q-value ≤0.1. Themultiple testing adjustment strategy with rejecting at 0.05 significancelevel and corresponding q<0.1 ensured that less than 10% of the findingsare false.

In a second analysis, the immunophenotype changes between pooledtreatment and placebo groups, where all three RAD001 dosing groups werecombined. To determine which immunophenotype changes differed betweenthe treated and placebo groups, within-patient cell count ratios foreach measured phenotype were calculated between baseline and Week 6 ofstudy drug treatment and between baseline and the end of study (Week12). The ratios were log transformed, and analyzed by analysis ofcovariance at each time point in order to detect a difference betweenthe pooled treatment and placebo groups. 152 tests in 76 phenotypes wereperformed to compare the pooled treatment effect against the placeboeffect. Stratified false discovery rate (FDR) control methodology wasimplemented to control the occurrence of false positives associated withmultiple testing yet provide considerably better power (Benjamini, Y. etal. (1995) J. Roy. Statist. 57:289-300; and Sun, L. et al. (2006) Genet.Epidemiol. 30:519-530). The cell type group was taken as thestratification factor and FDR (q-value) calculation was conducted withineach stratum respectively. All null-hypotheses at 0.05 significancelevel and q-value less than 20% were rejected. This can be interpretedas rejecting only those hypotheses with P values less than 0.05 and lessthan 20% probability that the each observed significant result is due tomultiple testing.

Results

In general, RAD001 was well tolerated, particularly the 0.5 mg daily and5 mg weekly dosing regimens. No deaths occurred during the study. Threesubjects experienced four serious adverse events (SAEs) that wereassessed as unrelated to RAD001. The 4 SAEs were retinal hemorrhage ofthe left eye with subsequent blindness in a subject with normal plateletcounts who had completed a 6 week course of 5 mg weekly RAD001 6 weekspreviously; severe back pain in a subject treated with placebo andsevere gastroenteritis in a subject treated with placebo. A list oftreatment-related adverse events (AEs) with an incidence >2% in anytreatment group is provided in Table 9. The most common RAD001-relatedAE was mouth ulcer that, in the majority of cases, was of mild severity.Overall, subjects who received RAD001 had a similar incidence of severeAEs as those treated with placebo. Only one severe AE was assessed asrelated to RAD001 mouth ulcers in a subject treated with 20 mg weeklyRAD001.

TABLE 9 Incidence of treatment-related AEs >2% in any treatment group bypreferred term RAD001 RAD001 RAD001 Placebo, 0.5 mg daily 5 mg weekly 20mg weekly pooled Total N = 53 n (%) N = 53 n (%) N = 53 n (%) N = 59 n(%) N = 218 n (%) Total AE(s) 35 46 109 21 211 Patients with AE(s) 22(41.5%) 20 (37.7%)  27 (50.9%) 12 (20.3%)  81 (37.2%) Mouth ulceration 6 (11.3%)  2 (3.8%)  9 (17.0%)  3 (5.1%)  20 (9.2%) Headache  0  2(3.8%)  9 (17.0%)  1 (1.7%)  12 (5.5%) Blood cholesterol  2 (3.8%)  2(3.8%)  2 (3.8%)  0  6 (2.8%) increased Diarrhea  1 (1.9%)  4 (7.5%)  1(1.9%)  0  6 (2.8%) Dyspepsia  0  3 (5.7%)  2 (3.8%)  1 (1.7%)  6 (2.8%)Fatigue  0  2 (3.8%)  4 (7.5%)  0  6 (2.8%) Low density lipoprotein  2(3.8%)  1 (1.9%)  2 (3.8%)  0  5 (2.3%) increased Tongue ulceration  3(5.7%)  1 (1.9%)  0  1 (1.7%)  5 (2.3%) Insomnia  1 (1.9%)  2 (3.8%)  1(1.9%)  0  4 (1.8%) Dry mouth  0  0  2 (3.8%)  1 (1.7%)  3 (1.4%)Neutropenia  0  0  3 (5.7%)  0  3 (1.4%) Oral pain  0  2 (3.8%)  1(1.9%)  0  3 (1.4%) Pruritus  0  2 (3.8%)  1 (1.9%)  0  3 (1.4%)Conjunctivitis  0  2 (3.8%)  0  0  2 (0.9%) Erythema  0  2 (3.8%)  0  0 2 (0.9%) Limb discomfort  0  2 (3.8%)  0  0  2 (0.9%) Mucosalinflammation  0  0  2 (3.8%)  0  2 (0.9%) Paresthesia oral  2 (3.8%)  0 0  0  2 (0.9%) Stomatitis  0  0  2 (3.8%)  0  2 (0.9%) Thrombocytopenia 0  0  2 (3.8%)  0  2 (0.9%) Urinary tract infection  0  0  2 (3.8%)  0 2 (0.9%)

The ability of RAD001 to improve immune function in elderly volunteerswas evaluated by measuring the serologic response to the 2012 seasonalinfluenza vaccine. The hemagglutination inhibition (HI) geometric meantiters (GMT) to each of the 3 influenza vaccine strains at baseline and4 weeks after influenza vaccination are provided in Table 10. Theprimary analysis variable was the HI GMT ratio (4 weeks postvaccination/baseline). The study was powered to be able to demonstratethat in at least 2 out of 3 influenza vaccine strains there was 1) a≥1.2-fold GMT increase relative to placebo; and 2) a posteriorprobability no lower than 80% that the placebo-corrected GMT ratioexceeded 1. This endpoint was chosen because a 1.2-fold increase in theinfluenza GMT ratio induced by the MF-59 vaccine adjuvant was associatedwith a decrease in influenza illness (Iob, A et al. (2005) EpidemiolInfect 133:687-693).

TABLE 10 HI GMTs for each influenza vaccine strain at baseline and at 4weeks after influenza vaccination RAD001 RAD001 RAD001 Influenza 0.5 mg5 mg 20 mg Vaccine daily weekly weekly Placebo Strain Time N = 50 N = 49N = 49 N = 55 A/H1N1 GMT (CV %) Baseline 102.8 84.2 90.1 103.2 (186.9)(236.4) (188.4) (219.7) Week 4 190.2 198.73 129.7 169.4 (236.9) (195.6)(175.9) (259.8) GMT ratio 2.6 2.5 1.8 2.0 (CV %) (302.5) (214.3) (201.5)(132.7) A/H3N2 GMT (CV %) Baseline 106.8 126.04 137.1 131.7 (168.2)(162.6) (211.5) (162.3) Week 4 194.4 223.0 223.0 184.3 (129.1) (118.8)(163.6) (153.2) GMT ratio 2.1 2.0 2.1 1.6 (CV %) (152.6) (189.2) (277.3)(153.6) B GMT (CV %) Baseline 44.2 64.8 58.0 57.0 (96.6) (87.3) (156.0)(112.6) Week 4 98.4 117.3 99.2 114.6 (94.8) (99.9) (124.1) (136.7) GMTratio 2.5 2.2 2.1 2.2 (CV %) (111.2) (112.8) (126.5) (109.2) Baselineindicates 2 weeks prior to influenza vaccination Week 4 indicates 4weeks after influenza vaccination N is number of subjects per cohort GMTis geometric mean titer GMT ratio is the GMT at week 4 postvaccination/GMT at baseline CV % indicates coefficient of variation

In the intent-to-treat (ITT) population, the low, immune enhancing, doseRAD001 (0.5 mg daily or 5 mg weekly) cohorts but not higher dose (20 mgweekly) cohort met the primary endpoint of the study (FIG. 33A). Thisdemonstrates that there is a distinct immunomodulatory mechanism ofRAD001 at the lower doses, and that at the higher dose the knownimmunosuppressive effects of mTOR inhibition may come into play.Furthermore, the results suggest a trend toward improved immune functionin the elderly after low, immune enhancing, dose RAD001 treatment.

In a subgroup analysis, the subset of subjects with low baselineinfluenza titers (≤1:40) experienced a greater RAD001-associatedincrease in titers than did the ITT population (FIG. 33B). These datashow that RAD001 is particularly effective at enhancing the influenzavaccine response of subjects who did not have protective (>1:40) titersat baseline, and therefore were at highest risk of influenza illness.

Scatter plots of RAD001 concentration versus increase in titer to eachinfluenza vaccine strain show an inverse exposure/response relationship(FIG. 34). Modeling and simulation based on mTOR mediatedphosphorylation of S6 kinase (S6K) predicts that the 20 mg weekly dosingregimen inhibits mTOR-mediated S6K activity almost completely, the 5 mgweekly dosing regimen inhibits S6K activity by over 50%, and the 0.5 mgdaily dosing regiment inhibits S6K phosphorylation by approximately 38%during the dosing interval (Tanaka, C et al. (2008) J. Clin. Oncol26:1596-1602). Thus, partial mTOR inhibition, e.g., mTOR-mediated S6Kphosphorylation, with low, immune enhancing, dose RAD001 may be as, ifnot more effective, than near complete mTOR inhibition with high doseRAD001 at enhancing the immune response of the elderly.

Rates of seroconversion 4 weeks after influenza vaccination were alsoevaluated. Seroconversion was defined as the change from a negativepre-vaccination titer (i.e., HI titer <1:10) to post-vaccination HItiter ≥1:40 or at least 4-fold increase from a non-negative (≥1:10)pre-vaccination HI titer. In the intention-to-treat population,seroconversion rates for the H3N2 and B strains were increased in theRAD001 as compared to the placebo cohorts although the increases did notmeet statistical significance (Table 11). In the subpopulation ofsubjects with baseline influenza titers <=1:40, RAD001 treatment alsoincreased the rates of seroconversion to the H3N2 and B strains, andthese results reached statistical significance for the B strain in the0.5 mg daily dosing cohort. These data further show that RAD001 enhancedthe serologic response to influenza vaccination in the elderly.

TABLE 11 Percent of subjects with seroconversion to influenza 4 weeksafter vaccination Placebo 0.5 mg 5 mg 20 mg N = 54 N = 48 N = 49 N = 48Intention to Treat Population H1N1 24 27 27 17 H3N2 17 27 24 25 B 17 2722 19 Subjects with Baseline Titers <=40 H1N1 40 42 45 36 H3N2 42 64 5371 B 16  40* 33 28 *Odds ratio for seroconversion between RAD001 andPlacebo significantly different than 1 (two-sided p-value <0.05 obtainedby logistic regression with treatment as fixed effect)

Current seasonal influenza vaccines often provide inadequate protectionagainst continuously emerging strains of influenza that present asvariants of previously circulating viruses. However, mice vaccinatedagainst influenza in the presence of the mTOR inhibitor rapamycin, ascompared to placebo, developed a broader serologic response toinfluenza. The broader serologic response included antibodies toconserved epitopes expressed by multiple subtypes of influenza thatprovided protection against infection with heterologous strains ofinfluenza not contained in the vaccine (Keating, R et al. (2013) NatImmunology 14:2166-2178). To determine if RAD001 broadened the serologicresponse to influenza in the elderly volunteers, HI titers to 2heterologous strains of influenza not contained in the influenza vaccine(A/H1N1 strain A/New Jersey/8/76 and A/H3N2 strain A/Victoria/361/11)were measured. The increase in the HI GMT ratios for the heterologousstrains was higher in the RAD001 as compared to placebo cohorts (FIG.35). In addition, seroconversion rates for the heterologous strains werehigher in the RAD001 as compared to placebo cohorts. The increase inseroconversion rates in the 5 and 20 mg weekly RAD001 dosing cohorts wasstatistically significant for the H3N2 heterologous strain (Table 12).The H3N2 seroconversion rate for the pooled RAD001 cohorts was 39%versus 20% for the placebo cohort (p=0.007). The results presentedherein suggest that mTOR inhibition broadens the serologic response ofelderly volunteers to influenza vaccination, and increases antibodytiters to heterologous strains of influenza not contained in theseasonal influenza vaccine.

Broadened serologic response to heterologous strains of influenza inmice treated with rapamycin has been associated with an inhibition ofclass switching in B cells and an increase in anti-influenza IgM levels(Keating, R. et al. (2013) Nat Immunol 14:2166-2178). However,inhibition of class switching may not be involved in the broadenedserologic response in humans treated with RAD001 because thepost-vaccination anti-influenza IgM and IgG levels did not differbetween RAD001 and placebo treated cohorts (FIG. 36).

TABLE 12 Percentage of subjects who seroconvert to heterologous strainsof influenza 4 weeks after seasonal influenza vaccination Placebo,RAD001 RAD001 RAD001 pooled 0.5 mg daily 5 mg weekly 20 mg weekly A/H1N1strain: 7% 17% 16% 8% A/NewJersey/8/76 A/H3N2 strain: 20% 38% 39%* 40% *A/Victoria/361/11 *Odds ratio for seroconversion between RAD001 andPlacebo significantly different than 1 (two-sided p-value < 0.05obtained by logistic regression with treatment as fixed effect)

To address the mechanism by which RAD001 enhanced immune function inelderly volunteers, immunophenotyping was performed on PBMC samplesobtained from subjects at baseline, after 6 weeks of study drugtreatment and 4 weeks after influenza vaccination (6 weeks after studydrug discontinuation). Although the percentage of most PBMC subsets didnot differ between the RAD001 and placebo cohorts, the percentage ofPD-1 positive CD4 and CD8 cells was lower in the RAD001 as compared toplacebo cohorts (FIG. 37). PD-1 positive CD4 and CD8 cells accumulatewith age and have defective responses to antigen stimulation becausePD-1 inhibits T cell receptor-induced T cell proliferation, cytokineproduction and cytolytic function (Lages, C S et al. (2010) Aging Cell9:785-798). There was an increase in percentage of PD-1 positive T cellsover time in the placebo cohort. At week 12 (4 weeks post-vaccination)this increase may have been due to influenza vaccination since influenzavirus has been shown to increase PD-1 positive T cells (Erikson, J J etal. (2012) JCI 122:2967-2982). However the percentage of CD4 PD-1positive T cells decreased from baseline at week 6 and 12 in all RAD001cohorts (FIG. 37A). The percentage of CD8 PD-1 positive cells alsodecreased from baseline at both week 6 and 12 in the two lower doseRAD001 cohorts (FIG. 37B). The percentage of PD-1 negative CD4 T cellswas evaluated and increased in the RAD001 cohorts as compared to theplacebo cohorts (FIG. 37C).

Under more stringent statistical analysis, where the results from theRAD001 cohorts were pooled and adjusted for differences in baseline PD-1expression, there was a statistically significant decrease of 30.2% inPD-1 positive CD4 T cells at week 6 in the pooled RAD cohort (n=84)compared to placebo cohort (n=25) with p=0.03 (q=0.13) (FIG. 38A). Thedecrease in PD-1 positive CD4 T cells at week 12 in the pooled RAD ascompared to the placebo cohort is 32.7% with p=0.05 (q=0.19). FIG. 38Bshows a statistically significant decrease of 37.4% in PD-1 positive CD8T cells at week 6 in the pooled RAD001 cohort (n=84) compared to placebocohort (n=25) with p=0.008 (q=0.07). The decrease in PD-1 positive CD8 Tcells at week 12 in the pooled RAD001 as compared to the placebo cohortis 41.4% with p=0.066 (q=0.21). Thus, the results from FIGS. 37 and 38together suggest that the RAD001-associated decrease in the percentageof PD-1 positive CD4 and CD8 T cells may contribute to enhanced immunefunction.

Conclusion

In conclusion, the data presented herein show that the mTOR inhibitorRAD001 ameliorates the age-related decline in immunological function ofthe human elderly as assessed by response to influenza vaccination, andthat this amelioration is obtained with an acceptable risk/benefitbalance. In a study of elderly mice, 6 weeks treatment with the mTORinhibitor rapamycin not only enhanced the response to influenzavaccination but also extended lifespan, suggesting that amelioration ofimmunosenescence may be a marker of a more broad effect on aging-relatedphenotypes.

Since RAD001 dosing was discontinued 2 weeks prior to vaccination, theimmune enhancing effects of RAD001 may be mediated by changes in arelevant cell population that persists after discontinuation of drugtreatment. The results presented herein show that RAD001 decreased thepercentage of exhausted PD-1 positive CD4 and CD8 T cells as compared toplacebo. PD-1 expression is induced by TCR signaling and remains high inthe setting of persistent antigen stimulation including chronic viralinfection. While not wishing to be bound by theory, is possible thatRAD001 reduced chronic immune activation in elderly volunteers andthereby led to a decrease in PD-1 expression. RAD001 may also directlyinhibit PD-1 expression as has been reported for the immunophilincyclosporine A (Oestreich, K J et al. (2008) J Immunol. 181:4832-4839).A RAD001-induced reduction in the percentage of PD-1 positive T cells islikely to improve the quality of T cell responses. This is consistentwith previous studies showing that mTOR inhibition improved the qualityof memory CD8 T cell response to vaccination in mice and primates(Araki, K et al. (2009) Nature 460:108-112). In aged mice, mTORinhibition has also been shown to increase the number of hematopoieticstem cells, leading to increased production of naïve lymphocytes (Chen,C et al. (2009) Sci Signal 2:ra75). Although significant differences inthe percentages of naïve lymphocytes in the RAD001 versus placebocohorts were not detected in this example, this possible mechanism maybe further investigated.

The mechanism by which RAD001 broadened the serologic response toheterologous strains of influenza may be further investigated. Rapamycinhas also been shown to inhibit class switching in B cells afterinfluenza vaccination. As a result, a unique repertoire ofanti-influenza antibodies was generated that promoted cross-strainprotection against lethal infection with influenza virus subtypes notcontained in the influenza vaccine (Keating, R et al. (2013) NatImmunol. 14:2166-2178). The results described herein did not show thatRAD001 altered B cell class switching in the elderly subjects who haddiscontinued RAD001 2 weeks prior to influenza vaccination. Although theunderlying mechanism requires further elucidation, the increasedserologic response to heterologous influenza strains described hereinmay confer enhanced protection to influenza illness in years when thereis a poor match between the seasonal vaccine and circulating strains ofinfluenza in the community.

The effect of RAD001 on influenza antibody titers was comparable to theeffect of the MF59 vaccine adjuvant that is approved to enhance theresponse of the elderly to influenza vaccination (Podda, A (2001)Vaccine 19:2673-2680). Therefore, RAD001-driven enhancement of theantibody response to influenza vaccination may translate into clinicalbenefit as demonstrated with MF59-adjuvanted influenza vaccine in theelderly (Job, A et al. (2005) Epidemiol Infect. 133:687-693). However,RAD001 is also used to suppress the immune response of organ transplantpatients. These seemingly paradoxical findings raise the possibilitythat the immunomodulatory effects of mTOR inhibitors may be dose and/orantigen-dependent (Ferrer, I R et al. (2010) J Immunol. 185:2004-2008).A trend toward an inverse RAD001 exposure/vaccination responserelationship was seen herein. It is possible that complete mTORinhibition suppresses immune function through the normalcyclophilin-rapamycin mechanism, whereas partial mTOR inhibition, atleast in the elderly, enhances immune function due to a distinctaging-related phenotype inhibition. Of interest, mTOR activity isincreased in a variety of tissues including hematopoietic stem cells inaging animal models (Chen C. et al. (2009) Sci Signal 2:ra75 and Barns,M. et al. (2014) Int J Biochem Cell Biol. 53:174-185). Thus, turningdown mTOR activity to levels seen in young tissue, as opposed to morecomplete suppression of mTOR activity, may be of clinical benefit inaging indications.

The safety profile of mTOR inhibitors such as RAD001 in the treatment ofaging-related indications has been of concern. The toxicity of RAD001 atdoses used in oncology or organ transplant indications includes rates ofstomatitis, diarrhea, nausea, cytopenias, hyperlipidemia, andhyperglycemia that would be unacceptable for many aging-relatedindications. However, these AEs are related to the trough levels ofRAD001 in blood. Therefore the RAD001 dosing regimens used in this studywere chosen to minimize trough levels. The average RAD001 trough levelsof the 0.5 mg daily, 5 mg weekly and 20 mg weekly dosing cohorts were0.9 ng/ml, below 0.3 ng/ml (the lower limit of quantification), and 0.7ng/ml, respectively. These trough levels are significantly lower thanthe trough levels associated with dosing regimens used in organtransplant and cancer patients. In addition, the limited 6 week courseof treatment decreased the risk of adverse events. These findingssuggest that the dosing regimens used in this study may have anacceptable risk/benefit for some conditions of the elderly. Nonetheless,significant numbers of subjects in the experiments describedhereindeveloped mouth ulcers even when dosed as low as 0.5 mg daily.Therefore the safety profile of low, immune enhancing, dose RAD001warrants further study. Development of mTOR inhibitors with cleanersafety profiles than currently available rapalogs may provide bettertherapeutic options in the future for aging-associated conditions.

Example 14: Enhancement of Immune Response to Vaccine in ElderlySubjects

Immune function declines in the elderly, leading to an increaseincidence of infection and a decreased response to vaccination. As afirst step in determining if mTOR inhibition has anti-aging effects inhumans, a randomized placebo-controlled trial was conducted to determineif the mTOR inhibitor RAD001 reverses the aging-related decline inimmune function as assessed by response to vaccination in elderlyvolunteers. In all cases, appropriate patent consents were obtained andthe study was approved by national health authorities.

The following 3 dosing regimens of RAD001 were used in the study:

20 mg weekly (trough level: 0.7 ng/ml)5 mg weekly (trough level was below detection limits)0.5 mg daily (trough level: 0.9 ng/ml)

These dosing regimens were chosen because they have lower trough levelsthan the doses of RAD001 approved for transplant and oncologyindications. Trough level is the lowest level of a drug in the body. Thetrough level of RAD001 associated with the 10 mg daily oncology dosingregimen is approximately 20 ng/ml. The trough level associated with the0.75-1.5 mg bid transplant dosing regimen is approximately 3 ng/ml. Incontrast, the trough level associated with the dosing regimens used inour immunization study were 3-20 fold lower.

Since RAD001-related AEs are associated with trough levels, the 3 dosingregimens were predicted to have adequate safety for normal volunteers.In addition, the 3 doses were predicted to give a range of mTORinhibition. P70 S6 Kinase (P70 S6K) is a downstream target that isphosphorylated by mTOR. Levels of P70 S6K phosphorylation serve as ameasure of mTOR activity. Based on modeling and simulation of P70 S6Kphosphorylation data obtained in preclinical and clinical studies ofRAD001, 20 mg weekly was predicted to almost fully inhibit mTOR activityfor a full week, whereas 5 mg weekly and 0.5 mg daily were predicted topartially inhibit mTOR activity.

Elderly volunteers >=65 years of age were randomized to one of the 3RAD001 treatment groups (50 subjects per arm) or placebo (20 subjectsper arm). Subjects were treated with study drug for 6 weeks, given a 2week break, and then received influenza (Aggrippal, Novartis) andpneumoccal (Pneumovax 23, Merck), vaccinations. Response to influenzavaccination was assessed by measuring the geometric mean titers (GMTs)by hemagglutination inhibition assay to the 3 influenza strains (H1N1,H3N2 and B influenza subtypes) in the influenza vaccine 4 weeks aftervaccination. The primary endpoints of the study were (1) safety andtolerability and (2) a 1.2 fold increase in influenza titers as comparedto placebo in ⅔ of the influenza vaccine strains 4 weeks aftervaccination. This endpoint was chosen because a 1.2 fold increase ininfluenza titers is associated with a decrease in influenza illness postvaccination, and therefore is clinically relevant. The 5 mg weekly and0.5 mg daily doses were well tolerated and unlike the 20 mg weekly dose,met the GMT primary endpoint (FIG. 33A). Not only did RAD001 improve theresponse to influenza vaccination, it also improved the response topneumococcal vaccination as compared to placebo in elderly volunteers.The pneumococcal vaccine contains antigens from 23 pneumococcalserotypes. Antibody titers to 7 of the serotypes were measured in oursubjects. Antibody titers to 6/7 serotypes were increased in all 3 RADcohorts compared to placebo.

The combined influenza and pneumococcal titer data suggest that partial(less than 80-100%) mTOR inhibition is more effective at reversing theaging-related decline in immune function than more complete mTORinhibition.

Example 15: Low Dose mTOR Inhibition Increases Energy and Exercise

In preclinical models, mTOR inhibition with the rapalog rapamycinincreases spontaneous physical activity in old mice (Wilkinson et al.Rapamycin slows aging in mice. (2012) Aging Cell; 11:675-82). Ofinterest, subjects in the 0.5 mg daily dosing cohort described inExample 2 also reported increased energy and exercise ability ascompared to placebo in questionnaires administered one year after dosing(FIG. 39). These data suggest that partial mTOR inhibition with rapalogsmay have beneficial effects on aging-related morbidity beyond justimmune function.

Example 16: P70 S6 Kinase Inhibition with RAD001

Modeling and simulation were performed to predict daily and weekly doseranges of RAD001 that are predicted to partially inhibit mTOR activity.As noted above, P70 S6K is phosphorylated by mTOR and is the downstreamtarget of mTOR that is most closely linked to aging because knockout ofP70 S6K increases lifespan. Therefore modeling was done of doses ofRAD001 that partially inhibit P70 S6K activity. Weekly dosing in therange of >=0.1 mg and <20 mg are predicted to achieve partial inhibitionof P70 S6K activity (FIG. 40).

For daily dosing, concentrations of RAD001 from 30 pM to 4 nM partiallyinhibited P70 S6K activity in cell lines (Table 13). These serumconcentrations are predicted to be achieved with doses of RAD001>=0.005mg to <1.5 mg daily.

TABLE 13 Percent inhibition of P70 S6K activity in HeLa cells in vitroRAD001 concentration 0 6 pM 32 pM 160 pM 800 pM 4 nM 20 nM % P70 S6K 0 018 16 62 90 95 inhibition

Conclusion

Methods of treating aging-related morbidity, or generally enhancing animmune response, with doses of mTOR inhibitors that only partiallyinhibit P70 S6K. The efficacy of partial mTOR inhibition with low dosesof RAD001 in aging indications is an unexpected finding. RAD001 doseranges between >=0.1 mg to <20 mg weekly and >=0.005 mg to <1.5 mg dailywill achieve partial mTOR inhibition and therefore are expected to haveefficacy in aging-related morbidity or in the enhancement of the immuneresponse.

Example 17: MSLN CART in Ovarian Cancer

Anti-tumor activity of a single dose of 2×10⁶ mesothelin CART cells waspreviously assessed in an in vivo ovarian tumor mouse model, asdescribed in Example 8. In order to see if the anti-tumor activity withthe M5 and M11 CAR T cells in this model, as described in Example 8,could be increased, the CAR T cells were administered a higher dose of4×10⁶ mesothelin CAR T cells 14 days after tumor implantation when thetumor volumes were an average of 150 mm³, and a follow-up identical doseof CART cells was administered. Half of the mice were given the secondidentical dose of the CAR T cells five days later to see if this wouldenhance the anti-tumor activity seen previously in this model.

Materials and Methods:

Cell line: OVCAR8 is a human ovarian cancer cell line that was derivedfrom a 64 year old female patient with ovarian adenocarcinoma, and hasbeen transduced to express mCherry. Cells were grown in RMPI mediumcontaining 10% fetal bovine serum. This cell line grows adherent intissue culture flasks. This cell line forms tumors in mice whenimplanted subcutaneously in the flank and mixed with matrigel. Theaddition of matrigel to the cells during the implantation allow for amatrix to develop in the flank of the mouse that provides a structurefor the tumor cells to start growing on. Over 10-12 days, the matrigelplug degrades and the solid tumor that remains is made up of tumor cellsand surrounding stromal cells. The OVCAR8 cells have been modified toexpress mCherry, so that that tumor cell growth can also be monitored byimaging the mice.

Mice: 6 week old NSG (NOD.Cg-Prkdc^(scid)Il2rg^(tm1Wjl)/SzJ) mice werereceived from the Jackson Laboratory (stock number 005557). Animals wereallowed to acclimate in the Novartis NIBRI animal facility for at least3 days prior to experimentation. Animals were handled in accordance withNovartis ACUC regulations and guidelines. Electronic transponders foranimal identification were implanted on the left flank one day prior totumor implantation. Mice were shaved on the right flank prior to tumorimplantation.

Tumor implantation: OVCAR8 cells in log phase were harvested aftertyrpsinization with 0.25% trypsin-EDTA. The cells were washed in 50 mlfalcon tubes at 1200 rpm for 5 minutes, once in growth media and thentwo times in cold sterile PBS. The cells were resuspended in PBS at aconcentration of 100×10⁶ per ml and kept on ice. The PBS solutioncontaining the cells was then mixed 1:1 with matrigel, resulting in afinal concentration of 50×10⁶ cells per ml of PBS-matrigel. The cellswere placed on ice and immediately implanted in mice. Tumors wereimplanted subcutaneously in 200 ul on the right flank.

The OVCAR8 model endogenously expresses mesothelin and thus, can be usedto test the in vivo efficacy of mesothelin directed CAR T cells. Thismodel grows well when implanted subcutaneously in the flank of mice andcan be calipered for tumor volume measurements. Upon implantation of10×10⁶ tumor cells in a 50:50 mix of PBS and matrigel, the tumorsestablish and can be accurately measured in one week. Within 13-15 days,the mean tumor volume is 100-200 mm³, with tumors reaching an endpointvolume (1000-1200 mm³) by 60-65 days. Anti-tumor activities oftherapeutic agents are often tested once tumors are fully engrafted andmatrigel resorbed. Thus, there is a large window with this model duringwhich the anti-tumor activity of the CAR T cells can be observed.

CAR T cell dosing: Mice were dosed with 4×10⁶ CART cells (13.3×10⁶ totalT cells) 14 days after tumor implantation. Cells were partially thawedin a 37 degree Celsius water bath and then completely thawed by theaddition of 1 ml of cold sterile PBS to the tube containing the cells.The thawed cells were transferred to a 15 ml falcon tube and adjusted toa final volume of 10 mls with PBS. The cells were washed twice at 1000rpm for 10 minutes each time and then counted on a hemocytometer. Tcells were then resuspended at a concentration of 133×10⁶ cells per mlof cold PBS and kept on ice until the mice were dosed. The mice wereinjected intravenously via the tail vein with 100 ul of the T cells fora dose of 4×10⁶ CAR T cells (13.3×10⁶ total T cells) per mouse. Sevenmice per group were either treated with 100 ul of PBS alone (PBS) or Tcells transduced with an isotype CAR (Isotype). 14 mice per group weretreated with either the SS1 mesothelin CAR T cells, M5 mesothelin CAR Tcells, or M11 mesothelin CART cells. Five days later the SS1, M5, andM11 groups were split in half and seven mice in each group were treatedwith an identical second dose of the CAR T cells. The groups wereidentified as SS1 single (one dose of SS1 CAR T cells), SS1 double (twodoses of SS1 CART cells), M5 single (one dose of the M5 CAR T cells), M5double (two doses of the M5 CAR T cells), M11 single (one dose of theM11 CAR T cells), M11 double (two doses of the M11 CAR T cells). Theisotype T cells, SS1 T cells, M5 T cells, and M11 T cells were allprepared from the same donor in parallel.

Animal monitoring: The health status of the mice was monitored daily,including twice weekly body weight measurements. The percent change inbody weight was calculated as(BW_(current)−BW_(initial))/(BW_(initial))×100%. Tumors were monitored2-3 times weekly by calipering and tumor volumes (TV) were calculatedusing the ellipsoid formula: TV(mm³)=((length×width²)×3.14159))/6. Tumorvolumes are reported as the mean+/− standard error of the mean (SEM).Percent treatment/control (T/C) values were calculated using thefollowing formula:

% T/C=100×ΔT/ΔC if ΔT≥0;

% Regression=100×ΔT/T _(initial) if ΔT<0;

where T=mean tumor volume of the drug-treated group on the final day ofthe study; T_(initial)=mean tumor volume of the drug-treated group oninitial day of dosing; ΔT=mean tumor volume of the drug-treated group onthe final day of the study−mean tumor volume of the drug treated groupon the initial day of dosing; C=mean tumor volume of the control groupon the final day of the study; and ΔC=mean tumor volume of the controlgroup on the final day of the study−mean tumor volume of the controlgroup on the initial day of dosing. T/C values in the range of 100%−42%are interpreted to have no or minimal anti-tumor activity; T/C valuesthat are ≤42% and >10% are interpreted to have anti-tumor activity ortumor growth inhibition. T/C values ≤10% or regression values ≥−10% areinterpreted to be tumor stasis. Regression values <−10% are reported asregression.

Results:

The anti-tumor activity of mesothelin CAR T cells were assessed in asubcutaneous ovarian adenocarcinoma xenograft model. Following tumorcell implantation on day 0, tumor bearing mice were randomized intotreatment groups and were administered 4×10⁶ CAR T cells (13.3×10⁶ totalT cells) intravenously via the lateral tail vein on day 14 after tumorimplantation. A second identical dose of T cells was given to half ofthe mice 5 days later. Tumor growth and animal health were monitoreduntil animals achieved endpoint. The mice in all groups were euthanizedbetween days 57 and 62 when tumors were beginning to show signs ofulceration leading to a change in animal body condition scores.

The mean+/−SEM tumor volume for all treatment groups is plotted in FIG.43. The PBS treatment group, which did not receive any T cells,demonstrates baseline OVCAR8 tumor growth kinetics in subcutaneouslyimplanted NSG mice. The Isotype treatment group received T cellstransduced with a control CAR. These cells serve as a T cell control toshow the non-specific response of human donor T cells in this model.Both the PBS and the Isotype treatment groups demonstrate continuoustumor progression throughout this study. The Isotype group shows aslight slower tumor growth, due to the background activity of the donorT cells. Similar to previous studies a single dose of 4×10⁶ CAR+ T cellsleads to no changes in tumor growth for the SS1 treated group. A doubledose of the SS1 CART cells similarly led to no changes in OVCAR8 tumorgrowth. The M5 and M11 single dose groups show clear anti-tumoractivity, as they did previously when dosed with 2×10⁶ CAR+ T cells.Both the M5 and M11 double dose groups show an increased anti-tumoractivity as compared with the single dose groups. The tumors in thesegroups show regression as opposed to tumor growth inhibition. The M5double dose group demonstrates a complete regression in four out ofseven mice with no tumor measureable for multiple time points.

Change in tumor volume versus the Isotype group (delta T/C %) wascalculated on day 52, which is the last tumor volume measurement beforemice were removed due to tumors ulcerating. Table 14 shows the delta T/Cvalues for each group. Both the SS1 single and double dose groups showno anti-tumor activity, similar to the PBS control treated group. The M5single dose group shows tumor growth inhibition with a delta T/C valueof 30.28%, while the M5 double dose group shows regression with aregression value of −63.90%. The M11 single dose group shows minimalanti-tumor activity with a delta T/C value of 52.91%, while the M11double dose group also demonstrates regression with a regression valueof −15.80%.

TABLE 14 Delta T/C (Treatment Tumor Volume/Control) Group Day Delta T/C(%) Regression (%) PBS 52 162.23 NA Isotype 52 100.00 NA SS1 Single 52172.05 NA SS1 Double 52 166.15 NA M5 Single 52 30.28 NA M5 Double 52 NA−63.90 M11 Single 52 52.91 NA M11 Double 52 NA −15.80

DISCUSSION

This study demonstrated that the mesothelin specific CAR T cells (M5 andM11), when dosed a second time five days after an initial T cell dose,are capable of leading to the regression of OVCAR8 tumors. This responsealong with the anti-tumor activity following a single dose of both theM5 and M11 CAR T cells are durable. In addition, four out of seven micein the M5 double dose group demonstrate a complete regression with notumor measureable by caliper or palpation.

As seen previously, e.g., in Example 8, a single dose of 2×10⁶ CAR Tcells of the M5 or M11 CAR T cells shows anti-tumor activity and whatappears to be stasis in the OVCAR8 xenograft model in NSG mice. In thisstudy, a single dose of 4×10⁶ M5 or M11 CART cells shows the sameresult. Compared to the Isotype CAR T cells or the SS1 CAR T cells, theM5 and the M11 groups show clear anti-tumor activity even as a singledose (FIG. 43). Administering a second dose of the SS1 CAR T cells doesnot show an increased anti-tumor response. However, administering asecond dose of the M5 or M11 CAR T cells results in an enhancedanti-tumor response and leads to regression of the tumors in both ofthese groups.

The increased anti-tumor activity of the M5 and M11 CAR T cells can beattributed to several mechanisms. The first is that a larger dose of CART cells achieves regression in the OVCAR8 model. Increasing the dose ofCART cells, e.g., to 8×10⁶ CART cells, e.g., in a single dose, mayincrease anti-tumor activity and result in regression. Another mechanismis that additional doses of CAR T cells improve anti-tumor activity andtumor regression. For example, the initial dose ay result in someanti-tumor activity due to the expansion of the CAR T cells and theproduction of cytokines by the T cells. Giving an additional dose, e.g.,a second dose five days later while the cells are still undergoingexpansion, may increase the activity of the cells due to the cytokinesalready being produced by the earlier administered CAR T cells.

In addition, previous studies have shown the infiltration of CART cellsinto the OVCAR8 tumors. Infiltration of CAR T cells is also studied inthe mice that have received a double dose of the CAR T cells todetermine if there is a greater infiltration of CD8⁺ (cytotoxic) Tcells. The persistence of the CAR T cells is also evaluated through ananalysis of the cells in the spleen and bone marrow of these mice.

Example 18: Epitope Mapping by Hydrogen-Deuterium Exhchange-MassSpectrometry

Hydrogen-deuterium exchange (HDX) mass spectrometry was performed topredict the regions of mesothelin that contribute to the epitopesrecognized by scFv constructs SS1 and M5. In HDX, the hydrogens of theamide backbone of a target protein are exchanged with deuterium.Interaction between the target protein and a binding protein, e.g., anantibody, “protects” regions of the target protein from solventaccessibility, thereby preventing hydrogen exchange at the interfacebetween the target protein and the binding protein. Accordingly, HDXmass spectrometry can be used to probe for mapping protein bindinginterfaces, e.g., predicting the epitope of an antibody.

As described in Example 2, analysis of scFv constructs M5, M11, M12,M14, M16, M17, M21, and M23 by SPR-based epitope binning against SS1indicated that M5 and M11 bind to a different epitope than SS1. To gaininsight into the regions of the human mesothelin that may be bound bySS1 and M5, HDX mass spectrometry was performed as follows.

Cloning of Expression Plasmids

A DNA fragment corresponding to amino acids Val 297-Gly 588 ofMesothelin (Uniprot Q13421) was cloned into a mammalian expressionvector using the restriction sites HindIII and EcoRI at the 5′ and 3′respectively. Subsequently, a secretion signal was inserted at aminoterminus corresponding to the amino acids METDTLLLWVLLLWVPGSTGDAAQPAASE(SEQ ID NO: 376). At the carboxyl terminus, a V5-His tag correspondingto the amino acids TRGSGKPIPNPLLGLDSTRTGHHHHHHHHHHHH (SEQ ID NO: 377)was also inserted. The three predicted glycosylation sites were mutated:N388Q, N496Q and N523Q. The final expressed sequence is provided below:

Glycosylation Deficient Mesothelin (296-588; N388Q, N496Q, N523Q)(SEQ ID NO: 378) METDTLLLWVLLLWVPGSTGDAAQPAASEVEKTACPSGKKAREIDESLIFYKKWELEACVDAALLATQMDRVNAIPFTYEQLDVLKHKLDELYPQGYPESVIQHLGYLFLKMSPEDIRKWQVTSLETLKALLEVNKGHEMSPQVATLIDRFVKGRGQLDKDTLDTLTAFYPGYLCSLSPEELSSVPPSSIWAVRPQDLDTCDPRQLDVLYPKARLAFQNMQGSEYFVKIQSFLGGAPTEDLKALSQQQVSMDLATFMKLRTDAVLPLTVAEVQKLLGPHVEGLKAEERHRPVRDWILRQRQDDLDTLGLGLQGTRGSGKPIPNPLLGLDSTRTGHHHHHHHHHHHH

The scFv corresponding to the M5 IgG (e.g., SEQ ID NO: 43) was similarlycloned into a mammalian expression vector with the N-terminal leaderMALPVTALLLPLALLLHAARP (SEQ ID NO: 379) and a C-terminal eight histidinepurification tag GSHHHHHHHH (SEQ ID NO: 380). The final expressedsequence is provided below:

(SEQ ID NO: 381) MALPVTALLLPLALLLHAARPQVQLVQSGAEVEKPGASVKVSCKASGYTFTDYYMHWVRQAPGQGLEWMGWINPNSGGTNYAQKFQGRVTMTRDTSISTAYMELSRLRSDDTAVYYCASGWDFDYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSDIVMTQSPSSLSASVGDRVTITCRASQSIRYYLSWYQQKPGKAPKLLIYTASILQNGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCLQTYTTPDFGPGTKVEIKGSHHHHHHHH

Protein Expression and Purification

Both the Mesothelin and scFv M5 were expressed under the control of aCMV promoter in the Expi293 expression system (Life Technologies). Themesothelin and scFv plasmids were co-transfected into 2L of Expi293cells at a density of 2.3×10⁶ cells/mL and viability >97% using 1 mg ofeach purified plasmid and 5 mg of polyethylenimine (PEI) in Opti-MEMmedia (Life Technologies). Mesothelin was also transfected alonefollowing a similar protocol but into 1L of cells using 1 mg of plasmidDNA and 2.5 mg of PEI. The transfection proceeded for 5 days and thenthe cells were harvested when the viability reached 80%.

Once the viability dropped to 80%, the cells and conditioned media werecollected and spun down at 4000×g for 10 minutes. The conditioned mediawas then filtered through a 0.22 μM filter to clear any debris and thenstirred overnight with Roche's cOmplete His-Tag Purification Resin at 4°C. A total of 3 mL of beads was added to the scFv-M5/Mesothelinconditioned media and 1.5 mL of beads was added to the Mesothelin alonemedia.

The following morning, the beads were loaded onto a Bio-Rad Econo-Columnand washed with 15 column volumes of 50 mM Tris.C1 pH=8.0, 300 mM NaCland then 8 column volumes of 50 mM Tris.C1 pH=8.0, 300 mM NaCl, 20 mMImidazole. The proteins were then eluted from the column with 50 mMTris.C1 pH=8.0, 300 mM NaCl, 250 mM Imidazole. The eluted sample wascollected and concentrated to 6 mL and loaded onto a Superdex 75 16/60column connected to an AKTAxpress (GE) in 20 mM HEPES pH=7.4, 150 mMNaCl. Elutions corresponding to the expected size of the mesothelin:M5complex or Mesothelin alone were collected and concentrated to 5 mg/mL.

Epitope Mapping by Hydrogen-Deuterium Exchange/Mass Spectrometry

Hydrogen-deuterium exchange (HDx) in combination with mass spectrometry(MS) (Woods V L, Hamuro Y (2001) High Resolution, High-Throughput AmideDeuterium Exchange-Mass Spectrometry (DXMS) Determination of ProteinBinding Site Structure and Dynamics: Utility in Pharmaceutical Design.J. Cell. Biochem. Supp.; 84(37): 89-98.) was used to map the putativebinding site of scFv antibodies M5 and SS1 on human mesothelin(296-588),referred to herein as hMSLN₂₉₆₋₅₈₈ (SEQ ID No 278). In HDx, exchangeableamide hydrogens of proteins are replaced by deuterium. This process issensitive to protein structure/dynamics and solvent accessibility and,therefore, able to report on locations that undergo a decrease indeuterium uptake upon ligand binding.

HDx/MS experiments were performed using methods similar to thosedescribed in the literature (Chalmers M J, Busby S A, Pascal B D, He Y,Hendrickson C L, Marshall A G, Griffin P R (2006) Probing protein ligandinteractions by automated hydrogen/deuterium exchange mass spectrometry.Anal. Chem.; 78(4): 1005-1014.Chalmers, 2006). The experiments wereperformed on a Waters H Dx/MS platform, which included a LEAPautosampler, nanoACQUITY UPLC, and Synapt G2 mass spectrometer. Thedeuterium buffer used to label the protein backbone of hMSLN₂₉₆₋₅₈₈ withdeuterium was 25 mM HEPES, 150 mM NaCl, pH 7.4; the overall percentageof deuterium in the solution was 94.5%. For hMSLN₂₉₆₋₅₈₈ deuteriumlabeling experiments in the absence of antibody, 400 pmol ofhMSLN₂₉₆₋₅₈₈, volume of 6.9 μl, was diluted using 100 μl of thedeuterium buffer for 15 minutes at 4° C. The labeling reaction was thenquenched with 100 μl of chilled quench buffer at 2° C. for three minutesfollowed by injected onto the LC-MS system for automated pepsindigestion and peptide analysis.

For hMSLN₂₉₆₋₅₈₈ deuterium labeling experiments in the presence of scFvM5, 400 pmol of hMSLN₂₉₆₋₅₈₈ co-expressed with M5, volume of 6.9 μl, wasdiluted using 100 μl of the deuterium buffer for 15 minutes at 4° C. Thelabeling reaction was then quenched with 100 μl of chilled quench bufferat 2° C. for three minutes followed by injected onto the LC-MS systemfor automated pepsin digestion and peptide analysis. For hMSLN₂₉₆₋₅₈₈deuterium labeling experiments in the presence of scFv SS1, 400 pmol ofhMSLN₂₉₆₋₅₈₈ is combined with 480 pmol of SS1 scFv. After incubation ofSS1 with hMSLN₂₉₆₋₅₈₈ for 30 minutes at 4° C., the total volume of 6.9μl was diluted using 100 μl of the deuterium buffer for 15 minutes at 4°C. The labeling reaction was then quenched with 100 μl of chilled quenchbuffer at 2° C. for three minutes, and then injected onto the LC-MSsystem for automated pepsin digestion and peptide analysis.

All deuterium exchange experiments were quenched using 0.5 M TCEP and 3M urea (pH=2.6). After quenching, the exchanged antigen was subjected toon-line pepsin digestion using a Poroszyme Immobilized Pepsin column(2.1×30 mm) at 12° C. followed by trapping on a Waters Vanguard HSS T3trapping column. Peptides were eluted from the trapping column andseparated on a Waters BEH C18 1×100 mm column (maintained at 1° C.) at aflow rate of 40 μl/min using a binary 8.4 minute gradient of 2 to 35% B(mobile phase A was 99.9% water and 0.1% formic acid; mobile phase B was99.9% acetonitrile and 0.1% formic acid).

Peptides from hMSLN₂₉₆₋₅₈₈ that were monitored by the deuterium exchangeexperiments are indicated in FIG. 44 (each bar represents a peptide).Over 80% coverage of hMSLN₂₉₆₋₅₈₈ was achieved.

For differential experiments between antibody (M5 or SS1) bound andunbound states it is informative to examine the difference in deuteriumuptake between the two states. In FIGS. 45A and 45B a negative valueindicates that the mesothelin-antibody complex undergoes less deuteriumuptake relative to mesothelin alone. A decrease in deuterium uptake canbe due to protection of the region from exchangeable deuterium orstabilization of the hydrogen bonding network. In contrast, a positivevalue indicates that the complex undergoes more deuterium uptakerelative to mesothelin alone. An increase in deuterium uptake can be dueto destabilization of hydrogen bonding networks (i.e. localizedunfolding of the protein).

Differential deuterium exchange between apo mesolethin and mesothelincomplexed with either SS1 or M5 were considered significant if: 1) ifthe difference is greater than 0.75 Da or, in cases, where thedifference is equal to or less than 0.75 Da, 2) the difference isgreater than 0.2 Da and statistically significant (p<0.01) when usingthe Tukey test. See, e.g., Houde D, Berkowitz S A, Engen J R (2010) TheUtility of Hydrogen/Deuterium Exchange Mass Spectrometry inBiopharmaceutical Comparability Studies. J. Pharma. Sci.; 100(6):2071-2086; Chalmers, M J, Pascal B D, Willis S, Zhang J, iturria S J,Dodge J A, Griffin P R (2011) Methods for the Analysis of High PrecisionDifferential Hydrogen Deuterium Exchange Data Int. J. Mass Spectrom.;302(1-3): 59-68.

FIG. 45A shows the differential deuterium uptake of the M5-mesothelincomplex (black bars) and the differential uptake of the SS1-mesothelincomplex (grey bars) from amino acid position 297 to 464 on mesothelin.Over this region binding of M5 to mesothelin caused no protection andthere is some very slight destabilization observed. In contrast, thebinding of SS1 to mesothelin caused significant protection in thefollowing peptides: 297-315, 315-322, 316-325, 337-346, 337-349,350-375, and 369-376. Protection in the peptides 297-315, 315-322,316-325, 337-346, 337-349 is consistent with the publishedcrystallography structure where residues E313, F317, K319, P343, andY346 form a significant part of the epitope (Ma J, Tang W K, Esser L,Pastan I, Xia D (2012) Recognition of Mesotehlin by the TherapueticAntibody MORAb-009 J. Biol. Chem.; (287) 40: 33123-33131.) FIG. 45Bshows the differential deuterium uptake for the amino acid positions458-586 on mesothelin. Over this region, binding of M5, causedsignificant protection in the following peptides: 481-490, 483-490,498-510, 501-507, 531-540, 532-540, 532-546, 537-546, 545-558, 546-569,547-560, 558-571, 561-572. By comparing the protection of overlappingpeptides, we deduce that the regions 485-490, 498-507, 532-537, and545-572 are significantly protected in the M5-mesothelin complex. Incontrast, the SS1-mesothelin complex does not contain any peptides inthe region 458-586 that are significantly protected using the criteriastated above.

FIG. 46 provides a summary of the protected regions for both the M5 andSS1 complex. These data indicate that M5 protects exclusively towardsthe C-terminal side of mesothelin, and that amino acid residues 485-490,498-507, 532-537, and 545-572 may contribute to the M5-mesothelininteraction. In contrast, SS1 protects exclusively towards theN-terminal side of mesothelin and there was no observed overlap in theregions protected by SS1 and those protected by M5. The observation oftwo distinct protection patterns for SS1 and M5 indicate that M5 likelybinds a distinct epitope from that bound by SS1. M11 is expected to bindto a similar region of MSLN as compared to M5. M11 and M5 have highhomology I both light and heavy chains including identical CDR3 regions.

EQUIVALENTS

The disclosures of each and every patent, patent application, andpublication cited herein are hereby incorporated herein by reference intheir entirety. While this invention has been disclosed with referenceto specific aspects, it is apparent that other aspects and variations ofthis invention may be devised by others skilled in the art withoutdeparting from the true spirit and scope of the invention. The appendedclaims are intended to be construed to include all such aspects andequivalent variations.

1.-110. (canceled)
 111. An isolated nucleic acid molecule encoding achimeric antigen receptor (CAR), wherein the CAR comprises: i) a humananti-mesothelin binding domain, ii) a transmembrane domain, and iii) anintracellular signaling domain, and wherein said anti-mesothelin bindingdomain comprises: (a) a light chain variable region comprising: a lightchain complementary determining region 1 (LC CDR1) comprising the aminoacid sequence of SEQ ID NO: 209; a light chain complementary determiningregion 2 (LC CDR2) comprising the amino acid sequence of SEQ ID NO: 233;and a light chain complementary determining region 3 (LC CDR3)comprising the amino acid sequence of SEQ ID NO: 257; and (b) a heavychain variable region comprising: a heavy chain complementarydetermining region 1 (HC CDR1) comprising the amino acid sequence of SEQID NO: 144; a heavy chain complementary determining region 2 (HC CDR2)comprising the amino acid sequence of SEQ ID NO: 162; and a heavy chaincomplementary determining region 3 (HC CDR3) comprising the amino acidsequence of SEQ ID NO:
 185. 112. The isolated nucleic acid molecule ofclaim 111, wherein the anti-mesothelin binding domain comprises theamino acid sequence of the light chain variable region of SEQ ID NO: 49;or an amino acid sequence with 95-99% identity to the amino acidsequence of the light chain variable region of SEQ ID NO:
 49. 113. Theisolated nucleic acid molecule of claim 111, wherein the anti-mesothelinbinding domain comprises the amino acid sequence of the heavy chainvariable region of SEQ ID NO: 49; or an amino acid sequence with 95-99%identity to the amino acid sequence of the heavy chain variable regionof SEQ ID NO:
 49. 114. The isolated nucleic acid molecule of claim 111,wherein the anti-mesothelin binding domain comprises the amino acidsequence of the light chain variable region of SEQ ID NO: 49; and theheavy chain variable region of SEQ ID NO:
 49. 115. The isolated nucleicacid molecule of claim 111, wherein the anti-mesothelin binding domainis a scFv.
 116. The isolated nucleic acid molecule of claim 111, whereina) the encoded anti-mesothelin binding domain comprises the sequence SEQID NO: 49, or a sequence with 95-99% identify to the amino acid sequenceSEQ ID NO: 49; or b) the isolated nucleic acid sequence encoding theanti-mesothelin binding domain comprises the sequence of SEQ ID NO: 97,or a sequence with 95-99% identify to the nucleic acid sequence of SEQID NO:
 97. 117. The isolated nucleic acid molecule of claim 111, whereinthe encoded CAR includes a transmembrane domain that comprises atransmembrane domain of a protein selected from the group consisting ofthe alpha, beta or zeta chain of the T-cell receptor, CD28, CD3 epsilon,CD45, CD4, CD5, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134,CD137 and CD154.
 118. The isolated nucleic acid molecule of claim 111,wherein the encoded anti-mesothelin binding domain is connected to thetransmembrane domain by a hinge region, wherein a) the encoded hingeregion comprises the amino acid sequence of SEQ ID NO:2, or the encodedhinge region comprises a sequence with 95-99% identity to the amino acidsequence of SEQ ID NO:2, or b) the encoded hinge region comprises theamino acid sequence encoded by the nucleic acid sequence of SEQ IDNO:13, or the encoded hinge region comprises an amino acid sequenceencoded by a sequence with 95-99% identity to the nucleic acid sequenceof SEQ ID NO:13; c) the encoded hinge region comprises the amino acidsequence of SEQ ID NO: 36, or the encoded hinge region comprises asequence with 95-99% identity to the amino acid sequence of SEQ IDNO:36; or d) the encoded hinge region comprises the amino acid sequenceencoded by the nucleic acid sequence of SEQ ID NO: 37, or the encodedhinge region comprises an amino acid sequence encoded by a sequence with95-99% identity to the nucleic acid sequence of SEQ ID NO:
 37. 119. Theisolated nucleic acid molecule of claim 111, wherein the encodedintracellular signaling domain comprises a costimulatory domain, whereinthe encoded costimulatory domain comprises a functional signaling domainderived from a protein selected from the group consisting of OX40, CD2,CD27, CD28, CD5, ICAM-1, LFA-1 (CD11a/CD18), ICOS (CD278), and 4-1BB(CD137).
 120. The isolated nucleic acid molecule of claim 111, whereinthe encoded intracellular signaling domain comprises a functionalsignaling domain of 4-1BB and/or a functional signaling domain of CD3zeta.
 121. The isolated nucleic acid molecule of claim 111, wherein theencoded intracellular signaling domain comprises (i) the amino acidsequence of SEQ ID NO: 7 and the amino acid sequence of SEQ ID NO:9 orSEQ ID NO:10; (ii) the amino acid sequence of SEQ ID NO:7, and an aminoacid sequence with 95-99% identity to the amino acid sequence of SEQ IDNO:9 or SEQ ID NO:10, or (iii) an amino acid sequence with 95-99%identity to the amino acid sequence of SEQ ID NO:7, and the amino acidsequence of SEQ ID NO:9 or SEQ ID NO:10.
 122. The isolated nucleic acidmolecule of claim 111, wherein the encoded intracellular signalingdomain comprises the amino acid sequence of SEQ ID NO:7 and the aminoacid sequence of SEQ ID NO: 9 or SEQ ID NO: 10, wherein the sequencescomprising the intracellular signaling domain are expressed in the sameframe and as a single polypeptide chain.
 123. The isolated nucleic acidmolecule of claim 111, wherein the nucleic acid sequence encoding theintracellular signaling domain comprises the nucleic acid sequence ofSEQ ID NO: 18, or a sequence with 95-99% identity thereof, and thenucleic acid sequence of SEQ ID NO:20 or SEQ ID NO:21, or a sequencewith 95-99% identity thereof.
 124. The isolated nucleic acid molecule ofclaim 111, further comprising a leader sequence, wherein the leadersequence comprises the amino acid sequence of SEQ ID NO:
 1. 125. Anisolated chimeric antigen receptor (CAR) molecule encoded by the nucleicacid molecule of claim
 111. 126. An isolated chimeric antigen receptor(CAR) molecule comprising: i) a human anti-mesothelin binding domain,ii) a transmembrane domain, and iii) an intracellular signaling domain,wherein the human anti-mesothelin binding domain comprises: (a) a lightchain variable region comprising: a light chain complementarydetermining region 1 (LC CDR1) comprising the amino acid sequence of SEQID NO: 209; a light chain complementary determining region 2 (LC CDR2)comprising the amino acid sequence of SEQ ID NO: 233; and a light chaincomplementary determining region 3 (LC CDR3) comprising the amino acidsequence of SEQ ID NO: 257; and (b) a heavy chain variable regioncomprising: a heavy chain complementary determining region 1 (HC CDR1)comprising the amino acid sequence of SEQ ID NO: 144; a heavy chaincomplementary determining region 2 (HC CDR2) comprising the amino acidsequence of SEQ ID NO: 162; and a heavy chain complementary determiningregion 3 (HC CDR3) comprising the amino acid sequence of SEQ ID NO: 185.127. The isolated CAR molecule of claim 126, wherein the anti-mesothelinbinding domain is a scFv.
 128. The isolated CAR molecule of claim 126,wherein the anti-mesothelin binding domain comprises: (i) the lightchain variable region of the amino acid sequence of SEQ ID NO:49, andthe heavy chain variable region of the amino acid sequence of SEQ ID NO:49; or (ii) a light chain variable region comprising an amino acidsequence with 95-99% identity with the amino acid sequence of SEQ ID NO:49; and a heavy chain variable region comprising an amino acid sequencewith 95-99% identity to the amino acid sequence of SEQ ID NO:
 49. 129.The isolated CAR molecule of claim 126, wherein the anti-mesothelinbinding domain comprises the sequence of SEQ ID NO: 49, or a sequencewith 95-99% identity thereof.
 130. The isolated CAR molecule of claim126, wherein the transmembrane domain comprises a transmembrane domainof a protein selected from the group consisting of the alpha, beta orzeta chain of the T-cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5,CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137 and CD154.131. The isolated CAR molecule of claim 126, wherein the humananti-mesothelin binding domain is connected to the transmembrane domainby a hinge region, wherein the hinge region comprises the amino acidsequence of SEQ ID NO:2 or SEQ ID NO:36, or a sequence with 95-99%identity thereof.
 132. The isolated CAR molecule of claim 126, whereinthe intracellular signaling domain comprises a costimulatory domain,wherein the costimulatory domain comprises a functional signaling domainof a protein selected from the group consisting of OX40, CD2, CD27,CD28, CD5, ICAM-1, LFA-1 (CD11a/CD18), CD278 (ICOS) and 4-1BB (CD137).133. The isolated CAR molecule of claim 126, wherein the costimulatorydomain comprises the amino acid sequence of SEQ ID NO: 7; or an aminoacid sequence with 95-99% identity to the amino acid sequence of SEQ IDNO:
 7. 134. The isolated CAR molecule of claim 126, wherein theintracellular signaling domain comprises a functional signaling domainof 4-1BB and a functional signaling domain of CD3 zeta.
 135. Theisolated CAR molecule of claim 134, wherein the intracellular signalingdomain comprises: (i) the amino acid sequence of SEQ ID NO: 7 and theamino acid sequence of SEQ ID NO:9; (ii) the amino acid sequence of SEQID NO:7 and the amino acid sequence of SEQ ID NO: 10; (iii) the aminoacid sequence of SEQ ID NO:7 and an amino acid sequence with 95-99%identity to the amino acid sequence of SEQ ID NO:9 or SEQ ID NO: 10; or(iv) an amino acid sequence with 95-99% identity to the amino acidsequence of SEQ ID NO:7 and the amino acid sequence of SEQ ID NO:9 orSEQ ID NO:10.
 136. The isolated CAR molecule of claim 135, wherein theintracellular signaling domain comprises the amino acid sequence of SEQID NO: 7 and the amino acid sequence of SEQ ID NO: 9 or SEQ ID NO: 10,wherein the sequences comprising the intracellular signaling domain areexpressed in the same frame and as a single polypeptide chain.
 137. Theisolated CAR molecule of claim 126, further comprising a leadersequence, wherein the leader sequence comprises the amino acid sequenceof SEQ ID NO: 1, or a sequence with 95-99% identity to the amino acidsequence of SEQ ID NO:
 1. 138. A human anti-mesothelin binding domaincomprising a light chain variable region comprising a light chaincomplementary determining region 1 (LC CDR1), a light chaincomplementary determining region 2 (LC CDR2), and a light chaincomplementary determining region 3 (LC CDR3) of the anti-mesothelinbinding domain sequence of SEQ ID NO: 49, and a heavy chain variableregion comprising a heavy chain complementary determining region 1 (HCCDR1), a heavy chain complementary determining region 2 (HC CDR2), and aheavy chain complementary determining region 3 (HC CDR3) of the humananti-mesothelin binding domain sequence of SEQ ID NO:
 49. 139. The humananti-mesothelin binding domain of claim 138, wherein the humananti-mesothelin binding domain: (a) is a scFv comprising the light chainand the heavy chain of the amino acid sequence of SEQ ID NO: 49; or (b)the human anti-mesothelin binding domain comprises: (b)(i) a light chainvariable region comprising an amino acid sequence having a sequence with95-99% identity with the amino acid sequence of SEQ ID NO: 49; and(b)(ii) a heavy chain variable region comprising an amino acid sequencehaving a sequence with 95-99% identity to the amino acid sequence of SEQID NO:
 49. 140. A vector comprising the nucleic acid molecule of claim111, wherein the vector is selected from the group consisting of a DNA,a RNA, a plasmid, a lentivirus vector, adenoviral vector, or aretrovirus vector.
 141. An immune effector cell comprising the nucleicacid molecule of claim
 111. 142. A method of making an immune effectorcell comprising transducing an immune effector cell with a vector ofclaim
 140. 143. A method of generating a population of RNA-engineeredcells comprising introducing an in vitro transcribed RNA or syntheticRNA into a cell, wherein the RNA comprises the nucleic acid molecule ofclaim
 111. 144. A method of providing an anti-cancer immunity in amammal comprising administering to the mammal an effective amount of acell comprising a CAR nucleic acid molecule of claim
 111. 145. A methodof treating a mammal having a disease or condition associated with cellsthat express mesothelin comprising administering to the mammal aneffective amount of a cell comprising a CAR nucleic acid molecule ofclaim
 111. 146. The method of claim 145, wherein the disease is a cancerselected from the group consisting of mesothelioma, malignant pleuralmesothelioma, non-small cell lung cancer, small cell lung cancer,squamous cell lung cancer, or large cell lung cancer, pancreatic cancer,pancreatic ductal adenocarcinoma, pancreatic metastatic cancer, ovariancancer, colorectal cancer and bladder cancer, or any combinationthereof.
 147. The cell of claim 141, further expressing a polypeptidethat comprises an extracellular portion of any of PD1, PD-L1, CEACAM,LAG3, CTLA4, VISTA, CD160, BTLA, LAIR1, TIM3, 2B4 or TIGIT, associatedwith a second polypeptide that comprises a costimulatory domain and/or aprimary signaling domain.
 148. The method of claim 145, wherein: thenucleic acid comprises an in vitro transcribed nucleic acid, and isintroduced into the cell, and the subject receives an initialadministration of the cells comprising the nucleic acid, and one or moresubsequent administrations of cells comprising the nucleic acid, whereinthe one or more subsequent administrations are administered less than 15days, after the previous administration.
 149. An isolated chimericantigen receptor (CAR) molecule comprising i) a human anti-mesothelinbinding domain, ii) a transmembrane domain, and iii) an intracellularsignaling domain, wherein the human anti-mesothelin binding domaincomprises: a light chain complementary determining region 1 (LC CDR1)comprising the amino acid sequence of SEQ ID NO: 209, a light chaincomplementary determining region 2 (LC CDR2) comprising the amino acidsequence of SEQ ID NO: 233, and a light chain complementary determiningregion 3 (LC CDR3) comprising the amino acid sequence of SEQ ID NO: 257,and a heavy chain complementary determining region 1 (HC CDR1)comprising the amino acid sequence of SEQ ID NO:144, a heavy chaincomplementary determining region 2 (HC CDR2) comprising the amino acidsequence of SEQ ID NO: 162, and a heavy chain complementary determiningregion 3 (HC CDR3) comprising the amino acid sequence of SEQ ID NO: 185,and wherein the CAR molecule comprises the amino acid sequence of SEQ IDNO: 73, or a sequence with 95-99% identity thereof.
 150. An isolatednucleic acid molecule encoding a chimeric antigen receptor (CAR),wherein the CAR comprises: i) a human anti-mesothelin binding domain,ii) a transmembrane domain, and iii) an intracellular signaling domain,and wherein said anti-mesothelin binding domain comprises: (a) a lightchain variable region comprising: a light chain complementarydetermining region 1 (LC CDR1) comprising the amino acid sequence of SEQID NO: 209; a light chain complementary determining region 2 (LC CDR2)comprising the amino acid sequence of SEQ ID NO: 233; and a light chaincomplementary determining region 3 (LC CDR3) comprising the amino acidsequence of SEQ ID NO: 257; and (b) a heavy chain variable regioncomprising: a heavy chain complementary determining region 1 (HC CDR1)comprising the amino acid sequence of SEQ ID NO:144; a heavy chaincomplementary determining region 2 (HC CDR2) comprising the amino acidsequence of SEQ ID NO: 162; and a heavy chain complementary determiningregion 3 (HC CDR3) comprising the amino acid sequence of SEQ ID NO: 185;and wherein the nucleic acid encoding the CAR comprises the nucleic acidsequence of SEQ ID NO: 121 or a sequence with 95-99% identity thereof.151. The isolated nucleic acid molecule of claim 150, wherein thenucleic acid encoding the CAR comprises the nucleic acid sequence of SEQID NO:
 121. 152. The isolated nucleic acid molecule of claim 150, whichencodes a CAR comprising the amino acid sequence of SEQ ID NO: 73, or asequence with 95-99% identity thereof.
 153. The isolated nucleic acidmolecule of claim 150, which encodes a CAR comprising the amino acidsequence of SEQ ID NO:
 73. 154. The isolated nucleic acid molecule ofclaim 111, wherein the encoded CAR comprises a transmembrane domain thatcomprises the amino acid sequence of SEQ ID NO: 6, or a transmembranedomain that comprises an amino acid sequence with 95-99% identity to theamino acid sequence of SEQ ID NO:6.
 155. The isolated nucleic acidmolecule of claim 111, wherein the sequence encoding the transmembranedomain comprises the nucleic acid sequence of SEQ ID NO: 17; or asequence with 95-99% identify to the nucleic acid sequence of SEQ IDNO:17.
 156. The isolated nucleic acid molecule of claim 111, wherein theencoded costimulatory domain comprises the amino acid sequence of SEQ IDNO: 7, or a sequence with 95-99% identity to the amino acid sequence ofSEQ ID NO:
 7. 157. The isolated nucleic acid molecule of claim 111,wherein the sequence encoding the costimulatory domain comprises thenucleic acid sequence of SEQ ID NO: 18, or a sequence with 95-99%identity to the nucleic acid sequence of SEQ ID NO:
 18. 158. Theisolated CAR molecule of claim 126, wherein the transmembrane domaincomprises the amino acid sequence of SEQ ID NO: 6, or a sequence with95-99% identity to the amino acid sequence of SEQ ID NO:
 6. 159. Themethod of claim 148, wherein the cell is a T cell or NK cell.